Organelle Biogenesis and Intracellular Lipid Transport in Eukaryotes

Transcription

1 MICROBIOLOGICAL REVIEWS, Dec. 1991, p Vol. 55, No /91/ $02.00/0 Copyright C 1991, American Society for Microbiology Organelle Biogenesis and Intracellular Lipid Transport in Eukaryotes DENNIS R. VOELKER Lord & Taylor Laboratory for Lung Biochemistry, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, Colorado INTRODUCTION MEMBRANE LIPID COMPOSITION AND ITS IMPLICATIONS METHODS OF STUDYING LIPID TRANSPORT Fluorescent Prob6s Spin-Labeled Probes Plasma Membrane Modification and Isolation Target Organelle Modification Phospholipid Transfer Proteins PHOSPHATIDYLCHOLINE TRANSPORT Synthesis and Intr?.Endoplasmic Reticulum Transport Transport to the Plgma Membrane Transport to the Mitochondria Transport of ExogWpous Phosphatidylcholine PHOSPHATIDYLETHIANOLAMINE TRANSPORT Synthesis and Intra-Endoplasmic Reticulum Transport Transport tb the Mitochondria Transport to the Plasma Membrane Transport of Exogenous Phosphatidylethanolamine Mitochondrial Export of Phosphatidylethanolamine PHOSPHATIDYLINOSITOL TRANSPORT Intramitochondrial Transport in Yeasts Phosphatidylinositol Transfer Protein Activity in Yeasts PHOSPHATIDYLSERINE TRANSPORT Synthesis and Intra-Endoplasmic Reticulum Transport Transport to the Mitochondria in Intact and Permeabilized Cells Phosphatidylserine Import into Isolated Mitochondria Phosphatidylserine Transport and Nonspecific Lipid Transfer Protein Activity Transport of Exogenous Phosphatidylserine SPHINGOLIPID TRANSPORT Intracellular Metabolism and Transport of Sphingolipids to the Plasma Membrane Transport and Metabolism of Exogenous Sphingolipids from the Plasma Membrane to the Cell Interior DIACYLGLYCEROL TRANSPORT CHOLESTEROL TRANSPORT Transport from the Endoplasmic Reticulum to the Plasma Membrane Transport to the Mitochondria Transport of Exogenous Cholesterol SUMMARY AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES INTRODUCTION which these components of biological membranes are assembled into mature structures. Unlike the protein components One of the hallmarks of eukaryotic cells is the diverse of membranes, lipids do not undergo chemical modifications array of intracellular organelles which carry out specialized that target them to specific membranes or conformational functions. These specialized compartments are usually char- changes that trap them within such membranes. This review acterized by a unique complement of structural proteins and will present some recent findings about intracellular lipid enzymes and often by a particular lipid composition. Al- movement within eukaryotic cells. though the distribution of specific enzymes is usually absolute with respect to certain organelles, the distribution of MEMBRANE LIPID COMPOSITION AND ITS lipids tends to vary only in the porportion of certain lipid IMPLICATIONS classes that occur within a given membrane. The observed differences in lipid composition among different organelles Many of the membranes of eukaryotes exhibit notable raise fundamental questions concerning the mechanisms by similarities in their lipid composition. The structures of the 543

2 544 VOELKER MICROBIOL. REV. GLYCEROLIPIDS X-Substituent Name -H Diacylglycerol - PO0 Phosphatidic Acid Ft2C-O-Ca -PO_-CH2CH2N+(CH3)3 Phosphatidylcholine H6CO-CRa I H2C-O-X HC-OCRb nc0 -CRb3 - Po-CH CH N+H3 22 Phosphatidylethanolamine SPHINGOLIPIDS N+H3 P&3- CH2CH CO2 -P03- C6 H6(OH)5 Phosphatidylserine HOCH-CH=CH-(CH2) -CH3 -H Coramide Phosphatidylinositol I -N-CRc -PO3-CH2CH2N4CH3)3 Sphingomyelin H2C- -X -GIc Glucosyl ceramide - Gic-Gal Lactosyl ceramide COMMON FATrY ACID SUBSTITUTIONS UTILIZED IN LIPID ANALOGS O H 0CH3 -C (CH2)5N NO2 -C (CH2½2,Cl N N-0 Fluorescent (NBD) Analogs Spin Labeled Analogs FIG. 1. General structural features of glycerolipids and sphingolipids. The major structural elements of the lipid species discussed in this article are shown. The designation Ra, Rb, and RC for the acyl groups is used for convenience to indicate that a large degree of heterogeneity in thd length and unsaturation of the hydrophobic moiety can occur. The underlined acyl groups are those that are usually replaced by the fluorescently labeled and spin-labeled fatty acid analogs. Several classes of lipids have been intentionally omitted from this list for simplicity. In general, the missing classes of lipids represent hydrophilic substitutions of the X moiety of both glycerolipids and sphingolipids. lipids discussed in this article are shown in Fig. 1. In both yeast (34) and animal (17, 29, 41) cells, the most abundant lipid is phosphatidylcholine, which usually accounts for 40 to 55% of the phospholipid. Phosphatidylethanolamine is usually second in abundance (15 to 30%), followed by phosphatidylinositol (10 to 15%) and phosphatidylserine (5 to 15%). Under certain growth conditions, total yeast membranes can contain up to 30%. phosphatidylinositol (34). Phosphatidic acid, bisphosphatidylglycerol, phosphatidylglycerol, and the polyphosphoinositides make up from 5% to less than 0.1% of total lipids. The sphingolipids (principally sphingomyelin and glycosylceramides in animal cells and phosphoinositol derivatives of ceramide in yeast cells) constitute another 5 to 10% of membrane lipids. Cholesterol (or ergosterol in yeast cells) is another component of cell membranes, accounting for 20 to 30% of the total cellular lipid. These lipid distributions are given for total cell membranes. When the lipid contents of individual organelles are determined, several additional facts become apparent. Lipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are found in the endoplasmic reticulum, mitochondria, nuclei, plasma membrane, and Golgi apparatus (17, 29, 41, 103). The ratios of these lipids to each other can vary significantly among the different organelles, but each major class is still represented. Other common classes of lipids can show greater degrees of segregation. Bisphosphatidylglycerol (cardiolipin) is essentially restricted to the inner mitochondrial membrane. In contrast, phosphatidylserine, cholesterol, and sphingomyelin appear to be unable to accumulate at the inner mitochondrial membrane, although this latter group of lipids is represented in various proportions in the other organelle systems mentioned above. Cholesterol and sphingolipids are more highly concentrated in the plasma membrane than in any other organelle. Within the context of a given membrane bilayer, another level of lipid diversity can be found. In certain membranes, the most notable of which is the plasma membrane, the distribution of phospholipids across the transverse plane of the bilayer is asymmetric (65, 100). Thus, the external leaflet

3 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 545 of the plasma membrane is enriched in phosphatidylcholine and sphingolipids, whereas phosphatidylethanolamine and phosphatidylserine tend to be enriched in the inner leaflet. These simple observations indicate a number of general principles about membrane structure and biogenesis with regard to lipids. (i) Biological membranes exhibit intermembrane heterogeneity. As described briefly above (and in more detail below), the lipid composition of different organelles can vary in subtle ways (by having different proportions of lipids) or in more dramatic ways (by the exclusion or inclusion of certain lipid classes). (ii) Biological membranes show intramembrane heterogeneity. No biological membrane has yet been described in yeast or animal cells which is composed solely of one class of lipid. (iii) The lipid components of membranes can be asymmetrically disposed across the transverse plane of the bilayer. The asymmetric distribution of enzymes and transport proteins in membranes is a well-appreciated phenomenon. Lipids can also be asymmetrically distributed across a given membrane, although this property is not true for all lipids or all membranes. (iv) The synthesis of the bulk of cellular lipids appears restricted to a few closely related membranes. The majority of glycerophospholipids synthesized in animal cells are made within the endoplasmic reticulum (9, 37, 94, 96). In Saccharomyces cerevisiae, the synthetic enzymes are found in both the endoplasmic reticulum and the outer mitochondrial membrane (48). The terminal steps in sphingolipid synthesis appear to be confined to the Golgi (28, 31, 36, 42, 67, 80). Thus, membrane biogenesis is not an autonomous process for most organelles. Therefore, specific transport phenomena are required for organelle assembly. The focus of this review will be upon the process of lipid transport within eukaryotic cells. This field of research is presently in its infancy, but the major questions to be answered are clear; they include the following. (i) What are the biochemical mechanisms by which lipids are transported from their site of biosynthesis to the multiplicity of organelles that characterize eukaryotic cells? (ii) Are the mechanisms for lipid transport for one type of organelle the same as for other organelles? For example, are the general mechanisms for moving phospholipids from the endoplasmic reticulum to the mitochondria and the plasma membrane the same or different? (iii) Are different classes of lipids transported to the same organelle by identical mechanisms? (iv) Are the lipid compositions that characterize a given organelle achieved by transport of specific lipids to the target organelle or by selective removal or degradation of lipids? (v) Are the transport of lipid and proteins to a given organelle independent or interdependent processes? Work in the past few years has begun to provide important information regarding many of these questions, but large gaps in our knowledge still remain. METHODS OF STUDYING LIPID TRANSPORT A major impediment to progress in the field of lipid transport has been the lack of a simple method for measuring the process in living cells. In the past several years, a number of divergent techniques have been developed that have enabled transport reactions to be studied without laborious subcellular fractionation procedures. The development of these methods has enabled the initial characterization of the transport processes to be made for most of the cellular lipids to selected organelles. Fluorescent Probes Pagano and colleagues have pioneered the use of fluorescent analogs of phospholipids and sphingolipids to study both cytologically and biochemically the movement of these molecules in living cells (68). The most widely used analogs contain a short-chain fatty acid (aminocaproic acid), to which is attached the fluorochrome 4-nitrobenzo-2-oxa-1,3- diazole (NBD). The NBD-derivatized fatty acid is used to replace one of the fatty acid moieties of glycerolipids (usually at the sn-2 position) or the fatty acid found in amide linkage in sphingolipids (Fig. 1). These analogs exhibit two important properties: they are intensely fluorescent, and they have slight water solubility and high hydrophobic partition coefficients that essentially allow them to be inserted into cell membranes from the bulk-fluid phase with very rapid kinetics. The fluorescence property enables one to examine the movement of these lipids at the level of a single cell. The partitioning property has both assets and liabilities. The major asset is that the partitioning coefficients allow the experimenter to pulse-label the plasma membrane of intact cells with high efficiency. The liability of the partitioning property is that most lipids do not exhibit this rapid partitioning and therefore some of the transport properties observed in intact cells after labeling of the surface membrane are unlikely to reflect those of native lipids. This problem is a minor one for the circumspect experimenter. The NBD analogs of phosphatidylcholine (84), phosphatidylethanolamine (59, 85), phosphatidylserine (59), phosphatidylinositol (87), phosphatidic acid (69, 70), diacylglycerol (69), sphingomyelin (46), glucosylceramide (99), and ceramide (54, 55) have been used to examine lipid transport phenomena in intact cells and have provided new and important information about these processes. Spin-Labeled Probes Devaux and coworkers have used spin-labeled analogs of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin to examine the transbilayer movement of these lipids in intact cells and isolated organelles (10, 35, 61). Like the NBD lipids described above, short-chain spin-labeled lipids readily partition into biological membranes. The structure of a commonly used spinlabeled fatty acid analog is shown in Fig. 1. In many cases these lipid analogs can be readily removed from accessible membrane compartments by washing with albumin at reduced temperatures. The spin-labeled analogs are bound by soluble albumin but do not undergo significant transbilayer movement at low temperature. By quantitating the amount of a spin-labeled lipid analog present in albumin-washed and unwashed membranes, it is possible to determine for a variety of experimental conditions the rate and extent of transbilayer lipid movement. This approach has permitted characterization of intramembrane lipid translocators in erythrocytes and subcellular membranes. Plasma Membrane Modification and Isolation Specific modification of plasma membrane lipids that face the external environment with membrane-impermeant reagents provides a method for identifying and sampling new populations of molecules that arrive at the membrane. The modification procedures introduce chemical changes in the lipids which enable the investigator to distinguish the altered lipids from the unaltered lipids by suitable chromatographic

4 546 VOELKER techniques. In experiments of this type one usually labels the lipid of interest with an appropriate precursor (such as [3H]acetate or ['4C]acetate for cholesterol and [3H]ethanolamine or ['4C]ethanolamine for phosphatidylethanolamine). The arrival of newly synthesized labeled cholesterol at the plasma membrane can be examined by treating fixed intact cells at reduced temperatures with cholesterol oxidase (13, 51) and measuring the appearance of radiolabel in cholestanone. Only radiolabeled cholesterol that is present in the external leaflet of the plasma membrane will be accessible to the cholesterol oxidase, which does not enter the cell. A similar approach has also been taken with chemical modifying reagents such as trinitrobenzenesulfonate (TNBS) to measure the arrival of newly synthesized phosphatidylethanolamine at the plasma membrane (83). Treatment of cells with TNBS at reduced temperatures can be used to selectively modify the pool of phosphatidylethanolamine in the outer leaflet of the cell membrane. The derivatized form of the lipid, trinitrophenylphosphatidylethanolamine, can be readily separated from phosphatidylethanolamine by thinlayer chromatography. By using an analogous approach, the oligosaccharide moiety of gangliosides can also be modified by oxidation with sodium periodate and then further derivatized with dinitrophenylhydrazine. This technique has been used to investigate the mechanism of transport of newly synthesized gangliosides to the plasma membrane (27). A variation of the plasma membrane modification strategy involves the use of liposomes composed of phosphatidylcholine added to culture media to trap cholesterol that is desorbed from the cell surface (58, 75, 76). When cholesterol is also present in the liposomes, an exchange of plasma membrane cholesterol with liposome cholesterol occurs without significant perturbation of the resident pool size of the cell surface cholesterol. Thus, by using radiolabeled precursors for plasma membrane cholesterol, such as [3H] acetate or [3H]cholesterol ester present in low-density lipoprotein (LDL) particles (this latter species of cholesterol must be processed via endocytosis and lysosomal degradation), one can use the desorption procedure to detect the appearance of radiolabeled cholesterol at the cell surface. Another approach used for measuring the appearance of newly synthesized (radiolabeled) lipids at the cell surface involves rapid and specific plasma membrane isolation techniques. This method uses cationic beads to bind the plasma membrane and then sonication and centrifugation to remove (intracellular) membrane components that fail to bind the beads (23). If the initial adsorption step is sufficient to saturate the beads with plasma membrane, this procedure yields highly enriched surface membrane preparations. This approach has been used to examine the transport of phosphatidylcholine (38) and cholesterol (23, 39, 89) to the cell surface. Target Organelle Modification Posttranslational modifications to proteins (such as removal of signal peptides, attachment of oligosaccharides, and sequential modifications to oligosaccharides) can be used to assess which compartments of the cell a protein has traversed. This same general method can be used to monitor the movement of a few lipids. Phosphatidylserine decarboxylase is an enzyme located at the inner mitochondrial membrane of eukaryotes (24, 96, 114). When phosphatidylserine arrives at the inner mitochondrial membrane, it is decarboxylated to form phosphatidylethanolamine, and these two lipids can easily be separated by thin-layer chromatography. Since the site of synthesis of phosphatidylserine in animal cells is primarily the endoplasmic reticulum (17, 37, 94), the decarboxylation of nascent phosphatidylserine can be used as a measure of the movement of this lipid from its site of synthesis to the mitochondria. A further adaptation of this approach can be used with hepatic cells that contain significant levels of phosphatidylethanolamine-n-methyltransferase. This enzyme is found primarily in the endoplasmic reticulum, and it converts phosphatidylethanolamine to phosphatidylcholine by the sequential addition of three methyl groups. The conversion of mitochondrial phosphatidylethanolamine (derived from phosphatidylserine) to its N-methyl derivatives or phosphatidylcholine can therefore be used as a measure of phosphatidylethanolamine transport from the mitochondria to the endoplasmic reticulum (91, 92). Phospholipid Transfer Proteins MICROBIOL. REV. In 1968 Wirtz and Zilversmit reported the properties of a protein capable of exchanging phospholipids between populations of membranes (111). The purified protein did not achieve net transfer of lipid mass, but rather the one-for-one exchange of lipid between the donor and acceptor membranes. Since this seminal observation, a large number of these proteins have been identified and purified and several have been sequenced. In animal cells these proteins fall into three groups (110). The first group is characterized by a high degree of specificity for phosphatidylcholine. The second group exhibits specificity for phosphatidylinositol but will also act on phosphatidylcholine, albeit with diminished kinetics. The third group will exchange virtually all lipids to some degree and is referred to as the nonspecific lipid transfer protein. This latter protein is identical to sterol carrier protein-2 (16, 63). In animal cells, in addition to phospholipid transfer proteins, there are also glycosylsphingolipid transfer proteins (1, 60). This latter family of proteins effects the translocation of a large range of glycosylsphingolipids from monoglycosylceramide to pentaglycosylceramides and gangliosides. This protein will not transfer glycerophospholipids between membranes. In Saccharomyces cerevisiae two types of phospholipid transfer proteins have been identified. One of these proteins exhibits greater specificity for phosphatidylinositol than for phosphatidylcholine (22). A second protein shows greatest specificity for phosphatidylcholine but will also act upon ethanolamine, serine, and inositol phospholipids (12). A basic problem that must be resolved with regard to the action of these proteins is whether they function in lipid transport processes in intact cells or whether the lipidbinding and lipid exchange property has some other purpose. Much of this debate arises from the observation that these proteins effect exchange reactions rather than net transport reactions in vitro. To date no investigator has devised a method for measuring the lipid transport activity of these proteins in intact cells. However, genetic experiments with yeast cells (2, 6) and biochemical analysis of mutant animal cell lines (97) have provided some new perspectives on this problem (see below).

5 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 547 Golgi Apparatus ChoP * PCho FIG. 2. Phosphatidylcholine transport processes. The translocation events for phosphatidylcholine within and among cellular membranes are shown. The symbol (s) represents the diacylglycerol moiety of phosphatidylcholine, and PCho is the standard abbreviation for the phosphocholine moiety. The asterisk denotes subcellular localizations that have been demonstrated in cells primarily with fluorescent analogs of phosphatidylcholine. Within the mitochondrial compartment the terms OM and IM refer to the outer and inner membranes, respectively. All rate constants are at temperatures of 37 C. PHOSPHATIDYLCHOLINE TRANSPORT Synthesis and Intra-Endoplasmic Reticulum Transport Phosphatidylcholine is the most abundant phospholipid present in animal cells and in S. cerevisiae (17, 29, 34, 41). This lipid is present in all cell membranes of these eukaryotes. The site of synthesis of phosphatidylcholine is primarily the endoplasmic reticulum in animal cells (17, 29, 37, 103) and both the endoplasmic reticulum and the outer mitochondrial membrane in yeast cells (48). The major transport processes for phosphatidylcholine are shown in Fig. 2. The enzymes responsible for the terminal steps of phosphatidylcholine synthesis in animal cells appear to have their catalytic sites at the cytosol-facing aspect of the endoplasmic reticulum (8). The newly synthesized lipid may therefore be initially inserted into the cytosolic face of the membrane. There is evidence for a transmembrane transporter that can translocate phosphatidylcholine or lysophosphatidylcholine to the luminal face of the endoplasmic reticulum (9, 40). The transporter is stereospecific, does not require ATP for its action, and is protease sensitive. It has been solubilized and reconstituted from rat liver microsomes but has not yet been purified (4). Additional experiments with a spin-labeled phosphatidylcholine analog (35) suggest the presence of a nonspecific transporter that is N-ethylmaleimide sensitive and acts upon a wide variety of glycerolipids. The t112 for translocation of spin-labeled phosphatidylcholine across the bilayer of the endoplasmic reticulum is 20 min. Transport to the Plasma Membrane The movement of newly synthesized phosphatidylcholine from its site of synthesis to the plasma membrane has been studied by Kaplan and Simoni using the rapid plasma membrane isolation procedure with Chinese hamster ovary (CHO-K1) cells (38). Nascent [3H]phosphatidylcholine was transported to the plasma membrane with a t12 of 1 min. The transport process was unaffected by the depletion of cellular ATP or the addition of colchicine, monensin, and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Furthermore, incubation of cells at 15 C had no significant effect upon the transport process. These results indicate that phosphatidylcholine movement to the plasma membrane can occur much faster than observed for protein transport. The energy independence of the process further distinguishes phosphatidylcholine transport from protein transport to the cell surface. The mechanism by which this rapid transport occurs remains unknown, but the data are consistent with the action of an energy-independent soluble carrier, such as phospholipid transfer proteins, that would rapidly equilibrate old and new populations of phosphatidylcholine among all cell membranes. Transport to the Mitochondria Newly made phosphatidylcholine is also rapidly transported from its site of synthesis to the outer mitochondrial membrane (112). The time required in vivo for radioequilibration between the endoplasmic reticulum-derived microsomes and the outer mitochondrial membrane is 5 min. The movement of labeled lipid in these experiments was investigated by pulse-chase experiments with [3H]choline followed by subcellular fractionation. In these studies the mitochondria were also subfractionated to yield inner and outer membrane components. Although the initial equilibration of phosphatidylcholine with the outer mitochondrial membrane was rapid, the equilibration of this lipid between the outer and inner membranes was comparatively slow. Following a 15-min pulse with [3H]choline, the ratio of specific radioactivities of outer and inner membrane phosphatidylcholine was 9.5. Unfortunately, in these studies no attempt was made to discern the mechanism of transport. However, the rapidity of the kinetics suggests that movement of nascent phosphatidylcholine to the plasma membrane and mitochondria may occur by similar processes. Transport of Exogenous Phosphatidylcholine The dynamics of the intracellular movement of phosphatidylcholine have also been examined by using fluorescent probes and Chinese hamster V79 fibroblasts (84). The lipid analog NBD-phosphatidylcholine can be readily and selectively inserted into the plasma membranes of cells at 2 C because of its hydrophobic partition coefficient. When the cells are maintained at 2 C the fluorescent lipid remains in the plasma membrane and can be readily removed from this membrane by simply washing the cells with a bathing solution containing an excess of liposomes that cause a

6 548 VOELKER "back-exchange" (reverse partitioning) to occur. The movement of the lipid out of the plasma membrane can be monitored by fluorescence microscopy, and the metabolism of the analog can also be measured by using conventional analytical techniques for lipids. When the fluorescently pulse-labeled cells are warmed to 37 C the NBD-phosphatidylcholine is observed to move from the plasma membrane to the Golgi apparatus (independently identified in the same cell by fluorescence microscopy with rhodamine-wheat germ agglutinin). The movement of the NBD-phosphatidylcholine to the Golgi can be blocked by depleting cells of ATP. If the cells are shifted to 16 C (rather than 37 C) after the initial labeling at 2 C, the fluorescent probe accumulates in an endosomal compartment. More detailed analysis of this process reveals that the NBD-phosphatidylcholine resides initially in the outer leaflet of the plasma membrane and subsequently in the luminal leaflet of the endosomal or Golgi membranes. The fluorescent lipid remains restricted to these organelle membrane leaflets, either because it cannot undergo transbilayer movement or because it does so extremely slowly. Slow transmembrane movement of phosphatidylcholine is observed not only for the NBD analog but also for native phosphatidylcholine present in phagosomal membranes (78). In contrast to the distribution of NBDphosphatidylcholine taken up by endocytosis, NBD-phosphatidylcholine that is microinjected into cells randomly labels all membranes (71, 84) as a consequence of its solubility and hydrophobic partitioning properties. These findings indicate that the endocytosed fluorescent phosphatidylcholine enters an intracellular compartment with restricted access to the cytosol. A combination of pulse-labeling and back-exchange procedures can be used to selectively label the Golgi apparatus with fluorescent phosphatidylcholine (84). After this labeling the movement of fluorescent lipid out of the Golgi and back to the cell surface can be monitored by fluorescence microscopy and endogenous phospholipase activity at the plasma membrane. By using this approach, Sleight and Pagano concluded that NBD-phosphatidylcholine movement out of the Golgi was a process that occurred with a t112 of 20 min. This rate of movement is similar to that observed for protein transport to the plasma membrane via vesicular intermediates. A survey of fluorescent phosphatidylcholine metabolism in a number of cultured cell lines reveals that the intracellular translocation of NBD-phosphatidylcholine may not follow the same pathway in all cell types (82). The exact reason for these differences among cell types is not known, but their existence indicates that some caution should be exercised in the application of this method to previously untested cell types. Thus, there is a marked discrepancy between the rates of movement of nascent phosphatidylcholine to the plasma membrane (t112 = 1 min) and the recycling of fluorescent phosphatidylcholine between the plasma membrane and the endosome and Golgi compartments (t112 = 20 min). The disparity in the results suggests that two entirely different processes are being studied. Of particular significance is the fact that the fluorescent lipid is segregated in membrane bilayer leaflets that do not interact with the cytosol. The exact location of the native phosphatidylcholine studied in the experiments by Kaplan and Simoni (38) is not known, but presumably the bulk of the lipid is located in the cytosol-facing pool of the endoplasmic reticulum immediately after its synthesis (8). These observations raise the intriguing possibility that phosphatidylcholines present in different leaflets of the plasma, Golgi, and endosomal mem- ATP MICROBIOL. REV. EtnP k_ - ATP, _ 7 9C ;,2 = 50 min FIG. 3. Phosphatidylethanolamine transport processes. The translocation events for phosphatidylethanolamine within and among cellular membranes are shown. Symbols and abbreviations are the same as for Fig. 1, and PEtn is the standard abbreviation for phosphoethanolamine. The term (fast) for phosphatidylethanolamine transport indicates that rate constants have not been determined but t1/2 values on the order of minutes are likely. All rate constants are at temperatures of 37 C. branes may experience markedly different intermembrane transport and equilibration rates. 0 PHOSPHATIDYLETHANOLAMINE TRANSPORT Synthesis and Intra-Endoplasmic Reticulum Transport Phosphatidylethanolamine can be synthesized in the mitochondria by the decarboxylation of phosphatidylserine (24, 96, 114) and in the endoplasmic reticulum and Golgi by the action of ethanolamine phosphotransferase (9, 37, 94), which utilizes diacylglycerol and CDP-ethanolamine as substrates. When cells are labeled with [3H]ethanolamine or [14C]ethanolamine, it is the latter pathway that gives rise to radiolabeling the phospholipid. The newly synthesized phosphatidylethanolamine made from an ethanolamine precursor appears to be made on the cytosolic face of the endoplasmic reticulum (8). Experiments with spin-labeled analogs of phosphatidylethanolamine indicate that this lipid is translocated from the cytosolic to the luminal leaflet of the endoplasmic reticulum with a t112 of 20 min (35). The major transport processes for phosphatidylethanolamine are shown in Fig. 3. Transport to the Mitochondria The transport of nascent phosphatidylethanolamine from its site of synthesis (primarily the endoplasmic reticulum) has been examined by using a [3H]ethanolamine precursor and subcellular frationation (112). Unlike the kinetics observed for phosphatidylcholine, the radioequilibration of newly made phosphatidylethanolamine between the endo- PEtn

7 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 549 plasmic reticulum and the mitochondria requires at least 2 h of chase with unlabeled precursor. The dependence of this transport phenomenon upon other metabolic processes or cytological structures has not been examined. Transport to the Plasma Membrane The movement of [3H]ethanolamine-labeled phosphatidylethanolamine from its site of synthesis (principally the endoplasmic reticulum) to the external leaflet of the plasma membrane has been measured by using chemical modification of the cell surface with TNBS (83). Under conditions where the TNBS is unable to enter the cell, the accumulation of radiolabel in the derivatized lipid (N-trinitrophenylphosphatidyl[3H]ethanolamine) can be used as a measure of the intracellular transport processes. In continuous-labeling experiments, [3H]phosphatidylethanolamine begins to arrive at the plasma membrane without a detectable lag, suggesting that transport of this lipid to the cell surface is a rapid event. In pulse-chase experiments, the [3H]phosphatidylethanolamine continues to arrive at the external leaflet of the plasma membrane for up to 2 h into the chase period, suggesting that although one component of the transport is fast, the complete equilibration between intracellular membranes and the outer leaflet of the plasma membrane is not as rapid. This may reflect equilibration rates between the inner and outer leaflets of the plasma membrane (see below). The kinetics of transport of phosphatidylethanolamine are markedly different from those observed for vesicle-based transport processes such as the secretion of proteins. Furthermore, metabolic inhibitors, such as monensin, that can disrupt protein secretion that occurs by vesicular mechanisms fail to alter the appearance of [3H]phosphatidylethanolamine at the cell surface. In addition, depletion of cellular ATP or treatment of cells with cytoskeletal poisons also has no effect upon phosphatidylethanolamine transport to the plasma membrane. Collectively, these results indicate that phosphatidylethanolamine synthesized at the endoplasmic reticulum is not transported to the cell surface via a vesicle-based mechanism. Additional evidence supporting this conclusion comes from studies with mitotic cells in which most vesicle traffic is arrested (44). Mitotic cells transport phosphatidylethanolamine (made from an ethanolamine precursor) to the plasma membrane at the same rate as interphase cells do. These results indicate that phosphatidylethanolamine transport from its site of de novo synthesis to the plasma membrane occurs via an ATP-independent and vesicle-independent mechanism. The precise nature of this transport process remains to be elucidated, but the results are consistent with a diffusion-based mechanism such as phospholipid transfer proteins. Transport of Exogenous Phosphatidylethanolamine The intracellular movement of phosphatidylethanolamine from the plasma membrane to the cell interior has been examined by using the NBD analog of this lipid (59, 85). The NBD-phosphatidylethanolamine can be readily inserted into the outer leaflet of the plasma membrane at 2 C. Upon warming of the cells to 37 C, the fluorescent lipid undergoes transbilayer movement and then appears rapidly in nuclear, mitochondrial, and Golgi membranes. The transbilayer movement can be prevented by depleting cells of ATP by using a combination of deoxyglucose and azide. The process does not occur at temperatures below 7 C. These latter results implicate the action of an ATP-dependent transporter in the mechanism. Additional evidence for the existence of an ATP-dependent phosphatidylethanolamine transporter has been obtained with erythrocytes. Using spin-labeled phosphatidylethanolamine analogs and analysis by electron spin resonance, Siegneuret and Devaux concluded that an ATPdependent aminophospholipid translocator was present in the plasma membrane (79). The t1/2 for translocation of spin-labeled phosphatidylethanolamine is 50 min, with an equilibrium distribution of 85 to 90% of this lipid located in the inner leaflet of the membrane (61). This approach has also been used to show that the translocation of spin-labeled phosphatidylethanolamine is bidirectional (10). Tilley et al. (86) also used erythrocytes to examine the transbilayer movement of phosphatidylethanolamine. Radiolabeled phosphatidylethanolamine was transferred to the erythrocyte membrane by the action of exogenous phospholipid exchange protein. Subsequently the cells were treated with phospholipase A2 and the accessibility of the inserted lipid to the enzyme was determined. The results indicated that cellular ATP was required for the time-dependent movement of phosphatidylethanolamine from the outer leaflet to the inner leaflet of the cell membrane. This transport protein (named the aminophospholipid translocator) has thus been independently identified by multiple criteria and shown to be present on both nucleated and nonnucleated cells. After NBD-phosphatidylethanolamine is translocated to the inner leaflet of the plasma membrane of Chinese hamster fibroblasts, it can be transferred into essentially all intracellular organelles primarily by its inherent hydrophobic partitioning properties. However, significant labeling of the Golgi apparatus does not occur at 7 C, even though the nuclear envelope and mitochondria become labeled at this temperature. In addition, modest depletion of cellular ATP levels (to 80% of the levels found in normal cells) does not alter the aminophospholipid translocator activity but selectively prevents labeling of the Golgi membranes (59, 85). These observations suggest that NBD-phosphatidylethanolamine may also gain access to the Golgi apparatus via endocytic mechanisms similar to those described for NBD-phosphatidylcholine. Mitochondrial Export of Phosphatidylethanolamine Additional transport phenomena for phosphatidylethanolamine have been described but not characterized in detail. Metabolic studies examining lipoprotein assembly in hepatocytes provide clear evidence that phosphatidylethanolamine synthesized in the mitochondria (by the decarboxylation of phosphatidylserine) is exported to the endoplasmic reticulum for either direct assembly into lipoproteins or further metabolism to form phosphatidylcholine (91) (see Fig. 4). The process of phosphatidylethanolamine export from the mitochondria also appears to occur in tissue culture cells (102) and yeast cells (21). Analysis of phosphatidylethanolamine precursor metabolism indicates that at least for several tissue culture cell lines, all of the requirements of the cell for phosphatidylethanolamine can be met by that made in the mitochondria by decarboxylation of phosphatidylserine (101). Recent studies also provide a direct demonstration that phosphatidylethanolamine generated within the mitochondria can be translocated to the plasma membrane with rapid kinetics. This transport process is unaffected by brefeldin A, a fungal metabolite that disrupts Golgi structure and function (93). These results clearly indicate that the mito-

8 550 VOELKER MICROBIOL. REV. chondria can export significant amounts of phospholipid to other organelles. PHOSPHATIDYLINOSITOL TRANSPORT Intramitochondrial Transport in Yeasts Intracellular transport of phosphatidylinositol in intact cells has not been directly determined. Isolated yeast mitochondria have been used to measure the movement of phosphatidylinositol from the outer to the inner mitochondrial membrane (81). Unlike animal cells, yeast cells appear to have phosphatidylinositol synthase in the outer mitochondrial membrane (48). Addition of [3H]inositol, CTP, and Mn2" to the mitochondria results in the synthesis of phosphatidyl[3h]inositol by phosphatidylinositol synthase acting upon endogenous substrates (81). This labeled pool of lipid is translocated from the outer to the inner mitochondrial membrane (as determined by the ability of phospholipid exchange protein to deplete the labeled mitochondria of newly synthesized 3H-labeled lipid). However, if the outer membrane pool of phosphatidylinositol is briefly pulselabeled and further synthesis of new lipid is arrested, the majority of the phosphatidylinositol remains in the outer membrane even with prolonged incubation. In addition, if phosphatidylinositol is inserted into the outer mitochondrial membrane by phospholipid exchange protein, it is not transported to the inner membrane. These observations suggest that phosphatidylinositol transfer from the outer to the inner membrane may be partially coupled to the synthesis of this lipid. Still other experiments (81) indicate that once phosphatidylinositol resides at the inner mitochondrial membrane, it does not move back to the outer membrane. Phosphatidylinositol Transfer Protein Activity in Yeasts Although the mechanism(s) by which phosphatidylinositol moves among organelles has not yet been determined, one potential process that has been the focus of much attention is the phosphatidylinositol transfer protein. Proteins exhibiting this activity have been identified in plants (3), animals (110), and yeasts (12, 22). Aitken et al. (2) purified the protein from S. cerevisiae and obtained primary-sequence information for the protein, which was then used to construct nucleic acid probes for screening a yeast genomic library. The gene for the phosphatidylinositol transfer protein (PIT) was subsequently cloned and sequenced. It was further used to make genetic constructs in which the wild-type gene for PIT was insertionally inactivated (with the selectable marker LEU2). By the use of standard yeast genetic methods, diploid strains carrying one active and one insertionally inactivated allele for PIT were constructed. When this diploid strain was induced to sporulate, no viable strains were obtainable from spores containing the inactive PIT gene. These results demonstrate that the PIT gene is an essential gene for S. cerevisiae. The sequence analysis for the yeast gene also revealed that this gene was identical to the SECI4 gene (6, 7). The corresponding secl4 mutants had been previously isolated as conditional (temperature-sensitive)-lethal mutants defective in protein secretion (7, 64). These mutants have the interesting property of accumulating large intracellular populations of Golgi membranes. It is not clear how the loss of phosphatidylinositol transfer protein relates to the accumulation of the Golgi, although there is speculation that lipid recycling between the Golgi and the endoplasmic reticulum is dis- 'A... ATP, 70C 2 =5 min FIG. 4. Phosphatidylserine transport processes. The transl8cation events for phosphatidylserine within and among cell membranes are shown. Symbols and abbreviations are the same as for Fig. 1 and 2, and PSer is the standard abbreviation for phosphoserine. The abbreviation psd represents the enzyme phosphatidylserine decarboxylase, and pemt represents phosphatidylethanolamine methyltransferase. Dashed lines denote processes which have not yet been defined but for which significant evidence exists. Phosphatidylethanolamine transport out of the mitochondria is shown in this figure, since this lipid can be made in the mitochondria by the decarboxylation of phosphatidylserine and the putative transport pathway has been examined only in this metabolic context. The term (fast) for phosphatidylserine transport between the outer and inner mitochondrial membrane indicates that rate constants have not been determined but t412 values on the order of minutes are likely. All rate constants are at temperatures of 37 C. rupted and that this leads to the observed increase. These exciting lines of experimentation have thus provided clear evidence for an important role for the phosphatidylinositol transfer protein in membrane dynamics in the yeast. A fundamental issue that still remains unresolved as these studies continue is whether the lipid exchange property of the protein seen in vitro is the basis for the action of this protein in vivo. PHOSPHATIDYLSERINE TRANSPORT Synthesis and Intra-Endoplasmic Reticulum Transport The interorganelle transport of phosphatidylserine has been examined by using whole cells, permeabilized cells, and cell-free systems with isolated organelles. The major subcellular site of phosphatidylserine synthesis appears to be the endoplasmic reticulum (37, 94). The major transport processes for phosphatidylserine are shown in Fig. 4. Spinlabeled analogs of phosphatidylserine introduced into the cytosolic face of microsomes readily undergo transbilayer movement to the inner leaflet with a t1/2 of 20 min (35), and it seems likely that this transport process also occurs in vivo. I I I I I

9 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 551 Transport to the Mitochondria in Intact and Permeabilized Cells The transport of newly synthesized phosphatidylserine to the mitochondria in intact cells has been studied by monitoring the kinetics of decarboxylation (102). The t112 for transport of phosphatidylserine to the mitochondria is 7 h if one assumes a single homogeneous pool. Bjerve (11) has, however, presented arguments that only a small fraction of the nascent phosphatidylserine pool is translocated to the mitochondria. The translocation of phosphatidylserine from its site of synthesis (principally the endoplasmic reticulum) to the mitochondria can be blocked by depleting cells of ATP (102). The phosphatidylserine accumulates in a subcellular fraction that is isolated as microsomes (which are derived largely but not exclusively from the endoplasmic reticulum). The results suggest that ATP is required for export of phosphatidylserine from the endoplasmic reticulum. Similar experiments performed by using yeast cells also suggest that the decarboxylation of nascent phosphatidylserine requires ATP (21). The ATP-dependent transport of phosphatidylserine can also be observed in saponin-permeabilized CHO-Kl cells, which are essentially devoid of all of their soluble proteins (104, 106). The transport process has been examined by using cells that have been pulse-labeled with [3H]serine (to label the phosphatidylserine pool) prior to the permeabilization step. The movement of the labeled phosphatidylserine to the mitochondria shows an absolute requirement for ATP. Neither AMP nor AMPPNHP (5'- adenylylimidodiphosphate) will support transport, and when apyrase (which sequentially dephosphorylates ATP to AMP) is added along with ATP, no transport occurs. The synthesis of phosphatidylserine can also occur in permeabilized cells, and this nascent phosphatidylserine is very efficiently translocated to the inner mitochondrial membrane (106). Labeling the phosphatidylserine pool after permeabilization has proved to be a useful approach for elucidating some of the basic characteristics of the transport process. The labeling of phosphatidylserine in permeabilized cells requires ATP and is functionally coupled to Ca2+ sequestration. By pulse-labeling the permeabilized cells with [3H]serine and then arresting Ca2+ sequestration [with ethylene glycol-bis(,-aminoethyl ether)-n,n,n',n'-tetraacetic acid (EGTA), ionomycin, or ditertiary butyl(hydro)quinone], it is possible to label the nascent pool of phosphatidylserine to high specific radioactivity and abruptly terminate further synthesis. Experiments such as these have provided clear evidence that transport of newly made phosphatidylserine to the mitochondria does not require ongoing synthesis of the lipid. The same conclusion is reached by arresting phosphatidylserine synthesis with ethanolamine, a Ca2+-independent competitive inhibitor of phosphatidylserine synthase. Although the transport of phosphatidylserine in permeabilized cells does not require continued synthesis of the lipid, it does require ATP. Depletion of ATP by the addition of apyrase efficiently arrests phosphatidylserine transport to the inner mitochondrial membrane. The t1/2 for transport of newly synthesized phosphatidylserine to the mitochondria in permeabilized cells is approximately 3 h if one assumes a single homogeneous pool of the lipid. Adriamycin, an antineoplastic drug that inhibits precursor protein import into the mitochondria, also inhibits phosphatidylserine import into this organelle in permeabilized cells (107). The site of action of adriamycin appears to be on phosphatidylserine translocation events between the outer and inner mitochondrial membrane. The effects of adriamycin upon both lipid and protein import into the mitochondria may be due to the complexing of the drug with bisphosphatidylglycerol present in the inner mitochondrial membrane and perturbation of membrane structure. Yet another property revealed by examining phosphatidylserine transport in permeabilized cells is the insensitivity of the process to dilution. If the phosphatidylserine pool is pulse-labeled with [3H]serine and then further synthesis is rapidly arrested by using EGTA, the effects of dilution upon the lipid transport process can readily be measured. A 45-fold dilution of the standard translocation reaction mixture containing permeabilized cells fails to yield any significant diminution of phosphatidylserine transport compared to the undiluted reaction mixture. This observation implicates a membrane-bound intermediate in the interorganelle transport process. These membrane-bound forms may be transport vesicles (that are unable to escape from the permeabilized cells) or may be confined to the endoplasmic reticulum or related membranes and translocated to the mitochondria via zones of adhesion or fusion. Phospliatidylserine Import into Isolated Mitochondria The import of phosphatidylserine into the mitochondria has been recohstituted with isolated orgahelles derived from rat liver (92, 105). The process is saturable with respect to the concentration of phosphatidylserine added as either microsomes or liposomes. The rate-limiting step in the process appears to be the transfer of the lipid from the donor membrane to the mitochondrial outer membrane. The ATP requirement for phosphatidylserine transport that is observed in intact and permeabilized cells has not been reconstituted with isolated organelles. Metabolic poisons that deplete mitochondrial ATP levels, uncouple oxidative phosphorylation, or deplete the electrochemical gradient do not alter the in vitro-reconstituted import of phosphatidylserine (105). This has led to speculation that the transport of phosphatidylserine to the mitochondria may be a two-step process consisting (in the cell) of an ATP-requiring step that places phosphatidylserine in a permissive environment that allows for the subsequent step involving ATP-independent transfer of the lipid to the outer mitochondrial membrane. Since the latter step does not require soluble proteins, it is thought to be a donor-acceptor membrane collision event. The exact nature of the membrane that donates phosphatidylserine to the mitochondria has not been clearly defined. Recent work by Vance (92) suggests that a unique population of membranes that normally sediment with rat liver mitochondria, but can be separated from them by centrifugation on Percoll gradients, transfers phosphatidylserine to the mitochondria with high efficiency in vitro. The marker enzyme profile of this membrane fraction is notably different from that of conventional microsomes. This membrane fraction exhibits high activity for phosphatidylserine synthase. In addition to facilitating transport of phosphatidylserine to the mitochondria, this fraction rapidly and efficiently metabolizes phosphatidylethanolamine, made in the mitochondria, to phosphatidylcholine by sequential methylation of the primary amine. These observations are consistent with the idea that restricted pools of phosphatidylserine may be shuttled to the mitochondria in liver cells and that when this lipid is decarboxylated, its product, phosphatidylethanolamine, is efficiently shuttled back to an endoplasmic reticulum-related membrane. Phosphatidylserine import into S. cerevisiae mitochondria has also been examined (81). Present evidence suggests that

10 552 VOELKER phosphatidylserine synthase is located in the outer mitochondrial membrane as well as the endoplasmic reticulum in S. cerevisiae (48). This topology of the enzymes of phosphatidylserine synthesis appears to distinguish yeast from animal cells. However, the possibility that specialized membranes might be closely associated with yeast mitochondria, as described above for liver cells, has not been rigorously ruled out. The synthesis of phosphatidylserine in yeast mitochondrial preparations can be partially driven by endogenous substrates with the addition of CTP, Mn2+, and [3H] serine (81). Phosphatidylserine synthesized in this manner is transported to the inner membrane and decarboxylated to form phosphatidylethanolamine. This transport does not require an electrochemical gradient across the inner membrane and most probably does not require ATP. The phosphatidylethanolamine made at the inner membrane is readily translocated back to the outer membrane. Although the biochemical details of these transport processes in both yeast and animal cells have not yet been elucidated, the overall properties seem quite similar in the two systems. Phosphatidylserine Transport and Nonspecific Lipid Transfer Protein Activity Several attempts have been made to correlate phosphatidylserine transport in the intact cell with phospholipid transfer protein activity. These investigations have focused upon the 14-kDa nonspecific lipid transfer protein. Under conditions where phosphatidylserine transport to the mitochondria is blocked by ATP depletion, the activity of the nonspecific lipid transfer protein is unaltered in cell extracts (102). The nonspecific transfer protein has been localized to peroxisomes by immunochemical methods (43, 88, 95). This protein has been shown to be essentially absent from liver tissue of patients with Zellweger syndrome, which is characterized by a dramatic deficiency of peroxisomes (90). These cells do contain an immunologically cross-reactive protein of 58 kda that can potentially serve as a precursor to the mature 14-kDa transfer protein (66). The mature nonspecific lipid transfer protein (but not the 58-kDa putative precursor) is also absent in mutant CHO-Kl cell lines that are defective in peroxisomal assembly (97). In these latter cells, phosphatidylserine transport to the mitochondria proceeds normally. These observations provide strong evidence against an obtigate role for the 14-kDa nonspecific lipid transfer proteih as the sole mechanism for phosphatidylserine transport between the endoplasmic reticulum and the mitochondria. Transport of Exogenous Phosphatidylserine In intact cells the plasma membrane has been pulselabeled with NBD-phosphatidylserine by incubation with the fluorescent lipid at 2 C (59). When the cells are warmed to 7 C the fluorescent lipid is rapidly internalized and labels essentially all intracellular membranes. The internalization of the NBD-phosphatidylserine is inhibited by depleting cellular ATP levels or by treating cells with N-ethylmaleimide or glutaraldehyde. In addition, the internalization of the NBD-phosphatidylserine is specific for the correct stereoisomer and is inhibited by the deacylated structural analogs glycerophosphoserine and glycerophosphoethanolamine. These findings are consistent with the conclusion that the internalization of the lipid is attributable to the action of the ATP-dependent aminophospholipid translocator (59, 79, 86) described above in the section on phosphatidylethanolamine transport. The transbilayer movement of phosphatidylserine in erythrocytes has also been described by using both spinlabeled lipid analogs (10, 79) and native species of the lipid (19, 20). Experimentation with the spin-labeled analogs has provided evidence that phosphatidylethanolamine and phosphatidylserine compete for the same lipid translocator and that the affinity of phosphatidylserine for this transporter is approximately 30 times the affinity of phosphatidylethanolamine (113). The t1/2 for spin-labeled phosphatidylserine transport across the bilayer of erythrocytes at 37 C is 5 min (113), and the process is bidirectional (10). The location of excess phosphatidylserine in either the extracellular or cytoplasmic domain of the erythrocyte can have profound effects upon the morphology of the cell (19, 20). When erythrocytes are exposed to unilamellar vesicles of dilauroyl phosphatidylserine, they spontaneously take up this lipid into the cell membrane. With excess phosphatidylserine in the outer monolayer, the cells become echinocytic. The rapid transport of phosphatidylserine into the inner bilayer via the action of the translocase causes the cells to temporarily resume normal morphology and then become markedly stomatocytic. Inhibition of the aminophospholipid translocase by treatment with diamide or N-ethylmaleimide blocks the transmembrane movement of phosphatidylserine and prevents the conversion of echinocytic cells to normal and then stomatocytic morphology. Removal of phosphatidylserine (that is restricted to the outer leaflet of the plasma membrane) from the sulfhydryl reagent-treated cells results in reversion to normal morphology. In addition to the transbilayer movement of phosphatidylserine, information has accumulated concerning the interorganelle movement of this lipid when supplied exogenously to cells. Transport processes for phosphatidylserine between the plasma membrane and the mitochondria have been inferred from studies with wild-type and mutant CHO-Kl cells based on the observation that extracellular phosphatidylserine is decarboxylated to form phosphatidylethanolamine (49, 62, 108). Because the inner mitochondrial membrane appears to be the sole subcellular location of phosphatidylserine decarboxylase (24, 94, 96, 114), the exogenous phosphatidylserine must have entered a pathway that transported it to the mitochondria. Virtually nothing is known about the intervening steps in this process. SPHINGOLIPID TRANSPORT MICROBIOL. REV. Intracellular Metabolism and Transport of Sphingolipids to the Plasma Membrane The major transport processes for sphingolipids are shown in Fig. 5. Work with fluorescent analogs of sphingolipids has been an important means of investigating the intracellular movement of these molecules. The initial studies with NBDceramide provided the first cytological evidence that sphingomyelin and glycosphingolipid synthesis was associated with the Golgi apparatus (54, 55). Subsequent studies involving detailed subcellular fractionation indicate that sphingomyelin synthesis occurs in the cisternae of the cis and medial Golgi (31, 36). When intact cells are exposed to NBDceramide at 2 C, it randomly partitions among all cell membranes (54, 55). Subsequent warming of the cells to 37 C leads to the synthesis of fluorescent sphingomyelin and glucosylceramide that are highly concentrated in the Golgi. Maintenance of the cells at 37 C allows transport of the

11 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 553 Glc -t 1-v sschop I Golgi Apparatus FIG. 5. Sphingolipid transport processes. The translocation events for mature sphingolipids among cell membranes are shown. The symbol (t-) represents the ceramide moiety of sphingolipids, Glc is the abbreviation for glucose, and PCho is the abbreviation for phosphocholine. The asterisk denotes subcellular localizations that have been demonstrated in cells primarily with fluorescent analogs of sphingolipids. For simplicity, the Golgi is shown as a single compartment. The site of sphingomyelin synthesis is now established as the cis and medial elements of the Golgi. The t1/2 value shown in heavy brackets applies to the overall process for internalization and recycling of sphingomyelin to the plasma membrane. All rate constants are at temperatures of 370C. nascent sphingolipids to the plasma membrane. The arrival of the mature sphingolipids at the outer leaflet of the plasma membrane can be determined by using back-exchange methods. The transport process has a t1/2 of 20 to 30 min, which is comparable to that observed for transport of proteins to the cell surface. Monensin, which blocks protein transport out of the Golgi, also blocks sphingolipid transport to the plasma membrane. These results are consistent with transport of sphingolipids and proteins in similar if not identical vesicles. To critically test whether vesicle movement was involved in sphingolipid transport, Kobayashi and Pagano (44) measured the process in mitotic cells. It is well established that protein transport from the Golgi to the plasma membrane is blocked during mitosis. These studies revealed that sphingolipid synthesis continues to occur in mitotic cells, but that transport of these molecules to the cell surface does not occur. These findings further substantiate a vesiclebased transport mechanism for newly synthesized NBDsphingolipids. The differential transport of sphingolipids between the apical and basolateral surfaces of the plasma membrane of epithelial cells has been investigated by van Meer et al. (99). The apical plasma membrane is relatively enriched in glucosylceramide compared with the basolateral membranes. This difference appears to be due in part to the kinetics of transport. The transport of NBD-glucosylceramide to the apical membrane proceeds at a rate two to three times higher that found for the basolateral membrane. These findings implicate a sorting mechanism for sphingolipids that most probably occurs at the level of the trans-golgi. The translocation of sphingomyelin synthesized from a ceramide analog containing an 8-carbon-chain fatty acid in amide linkage to the sphingosine moiety has recently been reported to occur in permeabilized Chinese hamster ovary cells (33). Following its synthesis in the Golgi, the nascent sphingomyelin analog is released from the cells into the media, almost certainly via vesicles that would normally be destined for the plasma membrane. The transport of these vesicles requires a cytosolic protein (that remains to be identified) and ATP. Secretion of the sphingomyelin analog does not occur at 15 C and is inhibited by GTP-yS [guanosine 5'-O-(3-thiotriphosphate)]. These findings have laid the groundwork for further biochemical dissection of the components involved in transporting sphingomyelin from the Golgi apparatus to the plasma membrane. The movement of gangliosides GDla, GM2, and GM3 to the plasma membrane of rat neuroblastoma and glioma cells has been monitored by using oxidation of the plasma membrane ganglioside with sodium periodate and later derivitization with dinitrophenylhydrazine (27). Like sphingomyelin and glucosylceramide, the time for transit was approximately 20 min. This transport was not examined for sensitivity to monensin. The synthesis of ganglioside GM3 from lactosylceramide, which requires lipid transport between Golgi compartments, has been reconstituted in a cell-free system by Wattenberg (109). The donor Golgi membranes are derived from Lec2 mutants of CHO cells that are defective in transport of CMP-sialic acid into the Golgi (26). These cells accumulate lactosylceramide. The acceptor Golgi is derived from Lec8 mutants of CHO cells that are defective in UDP-galactose transport and fail to make the lactosylceramide precursor for GM3 (25). In vitro mixing of the two Golgi populations with cofactors and [3H]CMP sialic acid demonstrates that the lactosylceramide in the Lec2 Golgi can be sialylated by the transferase present in the Lec8 Golgi. The process requires intact membranes, ATP, and cytosolic proteins. The lactosylceramide transport between Golgi membranes is kinetically similar to that for vesicular stomatitis virus (VSV) G protein. Both VSV G protein transport and lactosylceramide transport require an N-ethylmaleimide-sensitive factor and are inhibited by GTP-yS. These results indicate that glycolipid transport and glycoprotein transport through the Golgi are likely to be identical. Transport and Metabolism of Exogenous Sphingolipids from the Plasma Membrane to the Cell Interior Fluorescent sphingomyelin has also been used to study the movement of this lipid from the external leaflet of the plasma membrane to the cell interior (46). The NBD-sphingomyelin readily partitions into the cell surface membrane at 7 C. When the cells containing the fluorescent label are warmed to 37 C, the fluorescent sphingolipid is internalized and transported to the perinuclear region of the cell that colocalizes with centrioles. This transport process is disrupted by poisoning the cells with nocodazole. Nocodazole does not

12 554 VOELKER prevent internalization but leads to the accumulation of peripheral fluorescent vesicles and prevents the concentration of fluorescence in the perinuclear zone. This result implicates microtubule elements in the mechanism of assembling the fluorescent sphingomyelin into the centriolar region. The dynamics of NBD-sphingomyelin movement from the centriolar region to the cell surface have also been examined in living cells (46). This population of fluorescent sphingomyelin molecules is transported to the plasma membrane by a monensin-insensitive mechanism. The t112 for internalization and recycling back to the plasma membrane is 40 min. Further examination of the internalization and recycling of exogenously supplied NBD-sphingomyelin reveals that a small percentage of this lipid is also transported from the plasma membrane to lysosomes. Within the lysosomes this sphingomyelin is degraded to yield fluorescent ceramide, which subsequently enters the Golgi apparatus and generates a new population of fluorescent sphingomyelin molecules (46). The presence of this lysosomal targeting and processing pathway is made more evident in fibroblasts from patients with Niemann-Pick type A disease (47). These individuals are defective in lysosomal sphingomyelinase activity, and in their fibroblasts the exogenous NBD-sphingomyelin accumulates in the lysosomal fraction and cannot be degraded to ceramide. Using methods similar to that described for NBD-sphingomyelin, Kok et al. (45) have labeled the plasma membrane of BHK 21 cells with NBD-glucosylceramide. The glucosylceramide analog is endocytosed and colocalizes with endocytosed transferrin. Subsequently, the fluorescent glucosylceramide segregates into a vesicle population that does not contain transferrin. These vesicles may be identical to the pericentriolar vesicles described by Koval and Pagano (46). Ultimately, the glucosylceramide is recycled back to the plasma membrane. Collectively, the above studies of sphingolipid transport demonstrate several unique properties in the interorganelle movement of this lipid. The transport processes for newly synthesized mature sphingomyelin and its exogenously supplied counterpart are markedly different. These results suggest that highly selective sorting processes for lipids can occur at the plasma membrane as well as at intracellular membranes. DIACYLGLYCEROL TRANSPORT Insights into the transmembrane movement of diacylglycerol have been obtained from studies involving NBD-phosphatidic acid (69, 70) and NBD-phosphatidylinositol (87). Both of these fluorescent lipids are readily incorporated into the plasma membrane of cultured cells. In Chinese hamster V79 fibroblasts, NBD-phosphatidic acid is rapidly metabolized to NBD-diacylglycerol even at the low temperatures (2 C) used during the pulse-labeling of the plasma membrane (69, 70). This diacylglycerol is rapidly disseminated throughout the cell at 2 C. Studies with structural analogs of NBD-phosphatidic acid have clearly established that conversion to NBD-diacylglycerol is required before transmembrane movement and internalization can occur. Furthermore, NBD-phosphatidylcholine that has been introduced into the outer leaflet of the plasma membrane can be degraded by exogenous phospholipase C to yield NBDdiacylglycerol. This exogenous lipase treatment yields the same result as the generation of NBD-diacylglycerol catalyzed by plasma membrane phospholipases acting upon phosphatidic acid. The generation of NBD-diacylglycerol also occurs when Swiss 3T3 cells are incubated with NBDphosphatidylinositol at 7 C (87). The formation of NBDdiacylglycerol in plasma membrane in this system is accompanied by rapid transmembrane movement of the lipid and translocation throughout the cell interior to many organelles. These studies demonstrate that whenever NBD-diacylglycerol is formed in the plasma membrane, it rapidly undergoes transbilayer movement to the cytosolic face of the membrane. These findings are in excellent agreement with studies conducted by using model membranes and the thiol analog of diacylglycerol, which yields a t112 for transmembrane diacylglycerol movement of 15 s (32). The overriding implication of these studies is that the transbilayer movement of diacylglycerols derived from native membrane lipids is likely to be an extremely rapid process in biological membranes. One outstanding question of how the NBD-diacylglycerol is so rapidly translocated throughout the cell interior remains unresolved. Unlike its more polar phospholipid counterparts, NBD-diacylglycerol does not rapidly diffuse through the aqueous compartment. Thus, this fluorescent diacylglycerol must move either via an extremely efficient carrier (at 2 C) or by lateral diffusion across zones of intermembrane contact. CHOLESTEROL TRANSPORT MICROBIOL. REV. Transport from the Endoplasmic Reticulum to the Plasma Membrane Cholesterol is a major component of animal cell plasma membranes and is preferentially located in this membrane relative to intracellular membranes. The major pathways for cholesterol transport to the cell surface are shown in Fig. 6. Lange et al. (53) have reviewed existing data and analyzed the subcellular distribution of cholesterol by using density gradient methods coupled with marker enzyme analysis. They also used fluorescent-dye binding to estimate the cell surface area. The above studies have led the authors to the conclusion that in cultured human fibroblasts, as much as 90% of the total cellular cholesterol may reside in the plasma membrane. Using a morphometric approach, van Meer (98) has estimated that approximately 40% of the cellular cholesterol is found in the plasma membrane. The discrepancies between the two approaches remain unresolved, but either result demonstrates the marked enrichment of cholesterol in the plasma membrane. This cholesterol can be derived from de novo synthesis or recycling of lipoprotein-derived cholesterol. One method used to examine cholesterol transport to the plasma membrane has been to couple radioactive precursor labeling (primarily at the endoplasmic reticulum [77]), with rapid surface membrane isolation techniques (23, 39). These studies have revealed that after the terminal step in its synthesis, cholesterol moves to the cell surface with a t112 of 10 min. In the same experiments it was determined that the kinetics for cholesterol transport were clearly different from those observed for glycoprotein transport to the cell surface. Transport of cholesterol was inhibited by depletion of cellular ATP levels and was completely arrested at 15 C. The effects of metabolic poisons could be reversed in washout experiments, and the effects of 15 C incubation could be reversed by warming the cells to 37 C. The transport process was insensitive to cytoskeletal poisons or monensin, but maintenance of the cells at 15 C led to the accumulation of a low-density intracellular membrane fraction. The VSV G

13 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 555 \ATP [t,2 =1omin I the kinetics for translocation have a t112 of 1 h in the former and 10 min in the latter. It should be remembered, however, that these two approaches measure two different end point phenomena. The cholesterol oxidase method measures only cholesterol exposed at the external cell surface, whereas the rapid-isolation technique measures the labeled sterol found in the plasma and any associated membranes. Sequestration of cholesterol (e.g., in vesicles associated with but not contiguous with the cell surface) could easily account for the differences in the kinetics. Cholesterol Rich Vesicles Blocked In Nlemann- -(X - PlckTypeCorby U18666A IEsterification Regulation of HMG CoA Reductase, Membrane BIogeresIs ' LDL Receptor FIG. 6. Cholesterol transport processes. The translocation events for newly synthesized and LDL-derived cholesterol are shown. The t1/2 value in heavy brackets applies to the transport time between the endoplasmic reticulum and the plasma membrane. Details of the receptor-mediated endocytosis of LDL coupled to its receptor are well established and have been omitted from this figure for clarity. The question mark in the lower part of the figure emphasizes that the routes taken by LDL-derived cholesterol subsequent to its export from lysosomes are unknown. protein was observed to also accumulate in vesicles of similar density at 15 C when analyzed by density gradient centrifugation (89). Although the densities of the two populations of transport vesicles are similar, the populations appear to be involved in different transport processes. It is clear that at 15 C the VSV G protein accumulates in a pre-golgi compartment (determined by its sensitivity to endoglycosidase H). VSV G protein can also be made to accumulate in a pre-golgi compartment by poisoning the cells with brefeldin A, which causes the accumulation of proteins in the endoplasmic reticulum and disassembly of the Golgi apparatus (30). In contrast, brefeldin A has no effect on transport of cholesterol to the plasma membrane. These results demonstrate that cholesterol and VSV G protein (and, by inference, many plasma membrane proteins) travel to the cell surface via different vesicular intermediates. Experiments examining the arrival of nascent cholesterol at the plasma membrane, utilizing cholesterol oxidase to monitor the process, have yielded results consistent with those obtained by using the rapid plasma membrane isolation technique described above (51). Of particular importance is the independent identification by Lange and Steck (52) that a specialized membrane population, which they identify as the cholesterol-rich intracellular membrane fraction, functions as a transport intermediate in the movement of sterols to the plasma membrane. Some discrepancies remain in a few of the details obtained by the cholesterol oxidase analysis and the rapid plasma membrane analysis methods. In particular, Transport to the Mitochondria In addition to transport to the plasma membrane, newly synthesized cholesterol is transferred to the mitochondria in adrenal tissue. Although cholesterol is essentially excluded from the mitochondrial inner membrane in most tissues (103), in adrenal tissue it is transported to the inner mitochondrial membrane specifically for the synthesis of steroid hormones. This specialized process suggests that unique gene products involved in transporting sterols to the inner and outer mitochondrial membranes are expressed in steroidogenic tissue. A number of cellular components have been implicated in the process. Vinblastine and cytochalasin B inhibit the adrenocorticotropin-stimulated transport of newly synthesized cholesterol to the mitochondria in both isolated adrenal cells and in vivo studies, thereby showing that there is some role for microfilaments and microtubules in the process (18). The exact mechanisms by which cytoskeletal elements function in this system remain to be elucidated. The intramitochondrial transport of cholesterol can also be disrupted by using the agent cholesterol sulfate (50). Cholesterol sulfate blocks the metabolism of exogenous cholesterol by intact but not sonicated mitochondria. This inhibition has been taken as evidence for the presence of a regulated, tissue-specific cholesterol translocator in steroidogenic mitochondria. Additional in vitro studies (16) have also demonstrated that the nonspecific lipid transfer protein can enhance the delivery of cholesterol from lipid droplets to isolated mitochondria and that this cholesterol is then rapidly converted to pregnenolone. Transport of Exogenous Cholesterol Transport processes are present for exogenous as well as endogenous sources of cholesterol. The uptake and import of lipoprotein-associated cholesterol via high-affinity receptors is a well-understood and extensively studied phenomenon (14) that will not be reviewed in this article. However, the processes by which lipoprotein-derived cholesterol is transported out of the lysosomal compartment and subsequently utilized in membrane biogenesis, and regulatory phenomena associated with cholesterol metabolism, are only beginning to be addressed. Lipoprotein-derived cholesterol generated in the lysosomal compartment of fibroblasts normally has three functions or destinations: (i) transport to other organelles and use in membrane biogenesis, (ii) esterification and storage in the endoplasmic reticulum, and (iii) regulation of cholesterol biosynthesis (principally at the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase and LDL receptor expression). Brasaemle and Attie (13) have used cholesterol oxidase modification of cholesterol as a method of determining the rate of appearance of lipoproteinderived cholesterol at the plasma membrane. Their results indicate that once free cholesterol is formed within the lysosomal compartment (by hydrolysis of cholesterol ester),

14 556 VOELKER it is rapidly transported to the plasma membrane. The kinetics suggest a t1/2 for transport on the order of minutes. Individuals with the human genetic disorder Niemann- Pick type C disease accumulate significant amounts of cholesterol within their lysosomes (15). Detailed examination of sterol metabolism in fibroblasts from patients with Niemann- Pick type C disease (15, 56, 72-74) reveals that these cells are defective at negatively regulating cholesterol synthesis and LDL receptor activity and that they fail to stimulate cholesterol esterification in response to LDL-derived cholesterol. However, the biochemical machinery for regulating cholesterol synthesis, LDL receptor expression, and cholesterol esterification has been shown by independent criteria to be functional in these cells (56). On the basis of these observations, Liscum et al. (56, 58) have postulated that Niemann-Pick type C cells exhibit a defect in transporting LDL-derived cholesterol from the lysosomes to other compartments within the cell. To test this hypothesis, cells were incubated with LDL containing labeled cholesterol under conditions that permitted [3H]cholesterol accumulation within the lysosomal compartment (58). Subsequently the cells were washed and incubated in the presence of liposomes that would allow the exchange of plasma membrane cholesterol with liposomal cholesterol. This technique revealed that the mutant fibroblasts were markedly retarded in their ability to transport cholesterol from the lysosomal compartment to the plasma membrane when compared with normal fibroblasts. In striking contrast to this result, cholesterol synthesized de novo in the Niemann-Pick type C fibroblasts arrived at the plasma membrane with the same kinetics as observed for wild-type fibroblasts. This result provides strong circumstantial evidence that a defect in the mutant cells exists in the movement of cholesterol from lysosomes to the plasma membrane. At present, it is not known how many intervening cell compartments the cholesterol must traverse before arriving at the cell surface. Additional data supporting the hypothesis that the Niemann- Pick type C cells are defective in transport of lysosomal cholesterol to other compartments comes from the findings that the mutant cells (i) are inefficient at using LDL-derived cholesterol for membrane biogenesis, (ii) accumulate cholesterol in their lysosomal compartment to higher levels, and (iii) turn over lysosomal cholesterol more slowly than control cells do. Recently the drug U18666A (3-3-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to mimic the effects of the Niemann-Pick type C mutation in Chinese hamster ovary cells (57). This drug is likely to prove extremely valuable in further dissecting the biochemistry of intracellular cholesterol transport between lysosomes and other cellular compartments. SUMMARY AND FUTURE DIRECTIONS In a period of less than a decade, our understanding of the mechanisms involved in the interorganelle movement of lipids in eukaryotic cells has progressed from a point of almost complete ignorance to one in which some significant initial characterization of the processes involved for each major class of lipid between selected membranes has been accomplished. At present it is possible to clearly identify transport phenomena that are ATP dependent or independent, as well as those likely to be associated with vesicular intermediates. A striking feature of the observations thus far is the lack of a unifying mechanism for transport of different lipids. Vesicle-based transport processes encompass (i) ATP- MICROBIOL. REV. dependent phosphatidylcholine and sphingolipid export from the Golgi to the plasma membrane, (ii) ATP-dependent cholesterol transport from the endoplasmic reticulum to the plasma membrane, and (iii) ATP-dependent recycling of plasma membrane sphingomyelin and phosphatidylcholine. Differential susceptibility of the vesicular transport routes to monensin, GTP-yS, and brefeldin A indicates that movement of nascent cholesterol, of nascent sphingolipids, and of recycling pools of sphingomyelin and phosphatidylcholine proceed via different routes. Other ATP-dependent transport processes include (i) transmembrane movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and (ii) transport of phosphatidylserine from the endoplasmic reticulum to the mitochondria. The nature of the intermediates involved in phosphatidylserine translocation to the mitochondria remains to be elucidated. In contrast, ATP-independent lipid transport has been described for (i) transbilayer movement of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at the endoplasmic reticulum; (ii) phosphatidylethanolamine and phosphatidylcholine translocation from the endoplasmic reticulum to the plasma membrane; and (iii) phosphatidylserine translocation from the outer to the inner mitochondrial membrane. Superimposed upon all of these observations is the presence of phospholipid transfer proteins within all eukaryotic cells. The demonstration that phosphatidylinositol transfer protein is an essential gene in S. cerevisiae and belongs to the SEC gene family clearly proves that these proteins are involved in organelle assembly and turnover but still leaves open the issue of whether their principal function is lipid transport. The rapid kinetics of interorganelle movement of phosphatidylcholine from the endoplasmic reticulum to the mitochondria and plasma membrane is certainly consistent with a soluble carrier mechanism. Likewise, the rapid ATPindependent translocation of nascent phosphatidylethanolamine from the endoplasmic reticulum to the plasma membrane could be explained by a lipid transfer protein mechanism. Although rapid transport kinetics can occur via soluble carriers, this does not rule out other mechanisms such as permanent or temporary zones of continuity among organelles. The rapid distribution of diacylglycerol analogs within eukaryotic cells at 2 C suggests that zones of membrane apposition or continuity could permit either the collision-based transfer or lateral diffusion of certain lipid species. Although the details of lipid transport summarized above represent the major processes that have been elucidated thus far, there remain large deficiencies in what has been studied. There is virtually no information about lipid movement from the endoplasmic reticulum to the Golgi apparatus, lysosomes, and peroxisomes. The routes followed by phosphatidylinositol and phosphatidylserine from the endoplasmic reticulum to the plasma membrane remain almost entirely unexplored. Clearly, the major barrier to studying these transport processes is the lack of easily applied methods to measure them. Progress in this area requires the development of new technical approaches as well as genetic tools for dissecting these pathways. The transport of lipids from their site of synthesis to target organelles appears in many cases to be quite independent of protein transport. The pathways for nascent sphingolipid export and sphingolipid and phosphatidylcholine recycling from the plasma membrane conform with the general features of vesicle-based protein transport. However, most of

15 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 557 the other lipid trafficking appears to occur independently of protein transport routes. Intuitively this is problematic, since lipid must accompany membrane and secretory proteins in transport vesicles. Nonetheless, both kinetic criteria and inhibitor sensitivity indicate that newly synthesized cholesterol, phosphatidylcholine, and phosphatidylethanolamine destined for the plasma membrane can readily bypass the routes followed by membrane and secreted proteins between the endoplasmic reticulum and the plasma membrane or the external milieu. These results may indicate a segregation of lipid pools between newly synthesized molecules and those that are involved in membrane recycling and the vesicle-shuttling phenomenon. Even for sphingomyelin (which appears to follow the transit route for proteins from the Golgi to the plasma membrane) there is a clear segregation of the nascent pool produced in the Golgi and the pool that recycles to and from the plasma membrane (via pericentriolar vesicles). Future investigations of lipid trafficking must now begin to focus upon several major issues listed below. (i) What is the nature of transport vesicles? Vesicle intermediates in lipid transport have been identified in studies of cholesterol, sphingomyelin, and phosphatidylcholine translocation. It is important to understand the composition of these vesicles, particularly whether they contain unique populations of lipids and proteins. The isolation of these vesicles is a formidable task. Two outstanding problems are the acquisition of enough of the vesicles for analysis and the establishment of methods for assessing the purity of the vesicles. In some instances, favorable technological advances have already been made. For example, the transport vesicles involved in moving exogenous NBD-sphingomyelin and NBDphosphatidylcholine from the plasma membrane to the cell interior can be induced to accumulate by either nocadazole treatment or reduced temperature. As these vesicles contain fluorescent lipid trapped at the luminal leaflet of the membrane, the fluorescence, in principle, can be used as a specific marker for the vesicles. Provided that the integrity of the vesicles can be maintained during homogenization and fractionation procedures, this trapped fluorescent lipid should facilitate the identification and isolation of these transport vesicles. (ii) Are the de novo and recycling transport pathways different for all lipids? The striking differences in the timedependent distribution of nascent and exogenous sphingomyelin demonstrate that these two populations of lipids do not readily intermix within the cell. Similarly, the kinetics and inhibitor sensitivity for the distribution of nascent and exogenous phosphatidylcholine are so markedly different as to suggest that the two pools do not intermix. These differences for phosphatidylcholine may reflect the differences in transport and metabolism of lipid molecules at the cytosolic and the noncytosolic faces of cellular membranes. Do other lipids exhibit similar differences in their distribution depending upon whether they are newly synthesized or recycling? Does the capacity of a given lipid to undergo transbilayer movement at the plasma membrane ultimately determine whether it can freely mix with the nascent pool or whether it remains segregated in a recycling pool? These questions currently remain unanswered, and their resolution is likely to require the development of new methodology to address these issues. (iii) How is ATP utilized in transport phenomena? For vesicle-based processes, it is likely that ATP is utilized in moving vesicles along cytoskeletal elements. It is also likely that budding and fusion events among different membrane populations require ATP and GTP (and probably low-molecular-weight GTP-binding proteins) (5). The stochiometry of ATP utilization and the specific reactions that consume it in effecting lipid transport currently remain undefined. For ATP-requiring processes that have not yet been identified as being vesicle associated (such as the transport of nascent phosphatidylserine to the mitochondria), investigations must be carried out to determine whether vesicles are involved or whether some nonvesicular process is taking place. Serious consideration should be given to mechanisms of lipid transport that are not vesicular but ultimately require membrane movement and fusion. In cases in which different organelle membranes lie in close apposition (such as the endoplasmic reticulum and the mitochondria), it is possible that ATP is not used in the generation of vesicular intermediates, but rather is used in the formation of transient junctions or zones of continuity. (iv) What are the ATP-independent mechanisms of lipid transport? Two mechanisms seem most likely for ATPindependent transport: (i) zones of continuity among different membranes, and (ii) soluble carriers such as phospholipid transfer proteins. It should be emphasized that these possibilities need not be mutually exclusive. The results obtained with NBD-diacylglycerol movement within the cell at low temperature are very suggestive of lateral diffusion across regions of membrane continuity. However, if such continuities exist, there must be selective gating or filtering to prevent the randomization of lipid composition. The possibility that these putative intermembrane connections play a role in the rapid movement of nascent phosphatidylcholine and phosphatidylethanolamine cannot be ruled out. The dominant focus of research examining ATP-independent lipid transport will continue to be the phospholipid transfer proteins. The central question that remains is the following: do these proteins accomplish lipid transport in the intact cell? One approach that may prove useful in this area of research is the deliberate dissection of structure and function of the SEC14 gene product by site-directed mutagenesis to determine whether all mutations of the SEC14 protein that fail to support cell growth yield defective in vitro transport of phosphatidylinositol. Of special importance to the field of lipid transport will be the development of genetic tools to further dissect out the components of the process. As beautifully illustrated in the studies of the PITISEC14 gene, the ease of genetically manipulating S. cerevisiae makes it a prime eukaryotic system for addressing more refined questions about lipid translocation events. The development of rapid biochemical screens (or, ideally, selections) for isolating mutants of eukaryotic cells defective in lipid transport must be vigorously pursued. The acquisition of mutants with defects in lipid transport can be expected to greatly increase our understanding of these processes. Ultimately, a genetic approach to the problem will permit the characterization of the number of gene products involved in these phenomena. Complementation of transport mutants will enable the cloning of the genes involved and eventually yield their structure. ACKNOWLEDGMENTS I thank Peggy Hammond for excellent secretarial assistance. This work was supported by NIH grant GM REFERENCES 1. Abe, A., and T. Sasaki Purification and some properties of the glycolipid transfer protein from pig brain. J. Biol. Chem. 260:

16 558 VOELKER 2. Aitken, J., G. P. H. van Heusden, M. Temkin, and W. Dowhan The gene encoding the phosphatidylinositol transfer protein is essential for cell growth. J. Biol. Chem. 265: Arondel, V., and J. C. Kader Lipid transfer in plants. Experientia 46: Backer, J. M., and E. A. Dawidowicz Reconstitution of a phospholipid flippase from rat liver microsomes. Nature (London) 327: Balch, W. E Small GTP binding proteins in vesicular transport. Trends Biochem. Sci. 15: Bankaitis, V. A., J. R. Aitken, A. E. Cleves, and W. Dowhan An essential role for a phospholipid transfer protein in yeast Golgi function. Nature (London) 347: Bankaitis, V. A., D. E. Malehorn, S. D. Emr, and R. Greene The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108: Bell, R. M., L. M. Ballas, and R. A. Coleman Lipid topogenesis. J. Lipid Res. 22: Bishop, W. R., and R. M. Bell Assembly of phospholipids into cellular membranes: biosynthesis transmembrane movement and intracellular translocation. Annu. Rev. Cell Biol. 4: Bitbol, M., and P. F. Devaux Measurement of outward translocation of phospholipids across human erythrocyte membrane. Proc. Natl. Acad. Sci. USA 85: Bjerve, K. S The biosynthesis of phosphatidylserine and phosphatidylethanolamine from L-[3-14C]serine in isolated rat hepatocytes. Biochim. Biophys. Acta 833: Bozzato, R. P., and D. D. Tinker Purification and properties of two phospholipid transfer proteins from yeast. Biochem. Cell Biol. 65: Brasaemle, D. L., and A. D. Attie Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts. J. Lipid Res. 31: Brown, M. S., and J. L. Goldstein A receptor mediated pathway for cholesterol homeostasis. Science 232: Butler, J. D., M. E. Comly, H. S. Kruth, M. Vanier, M. Filling-Katz, J. Fink, N. Barton, H. Weintraub, J. M. Quirk, T. Tokoro, D. C. Marshall, R. 0. Brady, and P. G. Pentchev Niemann-Pick variant disorders: comparison of errors of cellular cholesterol homeostasis in group D and group C fibroblasts. Proc. Natl. Acad. Sci. USA 84: Chanderbhan, R., B. J. Noland, T. J. Scallen, and G. V. Vahouny Sterol carrier protein2. J. Biol. Chem. 257: Colbeau, A., J. Machbaur, and P. M. Vignais Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249: CriveUlo, J. F., and C. R. Jefcoate Intracellular movement of cholesterol in rat adrenal cells. J. Biol. Chem. 255: Daleke, D. L., and W. H. Heustis Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids. J. Cell Biol. 108: Daleke, D. L., and W. H. Huestis Incorporation and translocation of amino-phospholipids in human erythrocytes. Biochemistry 24: Daum, G., E. Heidorn, and F. Paltauf Intracellular transfer of phospholipids in the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 878: Daum, G., and F. Paltauf Phospholipid transfer in yeast. Isolation and partial characterization of a phospholipid transfer protein from yeast cytosol. Biochim. Biophys. Acta 794: DeGrella, R. F., and R. D. Simoni Intracellular transport of cholesterol to the plasma membrane. J. Biol. Chem. 257: Dennis, E. A., and E. P. Kennedy Intracellular sites of lipid synthesis and the biogenesis of mitochondria. J. Lipid Res. 13: MICROBIOL. REV. 25. Deutscher, S. L., and C. B. Hirschberg Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle me*nbranes. J. Biol. Chem. 261: Deutscher, S. L., N1 Nuwayhid, P. Stanley, E. I. Briles, and C. B. Hirschberg Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMPsialic acid transport. Cell 39: Dower, S., H. Miller-Podraza, and P. H. Fishman Translocation of newly synthesized gangliosides to the cell surface. Biochemistry 21: Eppler, C. M., D. J. Morre, and T. W. Keenan Ganglioside biosynthesis in rat liver: chdracterization of cytidine-5'- monophospho-n-acetylneuraminic acid:hematoside(gm3)salyltransferase. Biochim. Biophys. Acta 619: Esko, J. D., and C. R. H. Raetz Synthesis of phospholipids in animal cells, p In P. D. Boyer (ed.), The enzymes. Academic Press, Inc., New York. 30. Fujiwara, T., 0. Kimmimitsu, S. Yokota, A. Takatsuki, and Y. Ikehara Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J. Biol. Chem. 263: Futerman, A. H., B. Stieger, A. L. Hubbard, and R. E. Pagano Sphingomyelin synthesis in rat liver occurs predominantly at the cis and media cisternae of the Golgi apparatus. J. Biol. Chem. 265: Ganong, B. R., and R. M. Bell Transmembrane moveihent of phosphatidylglycerol and diacylglycerol sulfhydryl analogues. Biochemistry 23: Helms, J. B., A. Karrenbauer, K. W. A. Wirtz, J. E. Rothman, and F. T. Wieland Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. J. Biol. Chem. 265: Henry, S. A Membrane lipids of yeast: biochemical and genetic studies, p In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 35. Herrman, A., A. Zachowski, and P. F. Devaux Protein mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity. Biochemistry 29: Jeckel, D., A. Karrenbauer, R. Birk, R. R. Schmidt, and F. Wieland Sphingomyelin is synthesized in the Cis-Golgi. FEBS Lett. 261: Jelsema, C. L., and D. J. Morre Distribution of phospholipid biosynthetic enzymes among cell components of rat liver. J. Biol. Chem. 253: Kaplan, M. R., and R. D. Simoni Intracellular transport of phosphatidylcholine to the plasma membrane. J. Cell Biol. 101: Kaplan, M. R., and R. D. Simoni Transport of cholesterol from the endoplasmic reticulum to the plasma membrane. J. Cell Biol. 101: Kawashima, Y., and R. M. Bell Assembly of the endoplasmic reticulum bilayer. Transporter for phosphatidylcholine and metabolites. J. Biol. Chem. 262: Keenan, T. W., and D. J. Moore Phospholipid class and fatty acid composition of Golgi apparatus isolated from rat liver and comparison with other cell fractions. Biochemistry 9: Keenan, T. W., D. J. Moore, and S. Basu Ganglioside biosynthesis: concentration of glycosphingolipid glycosyltransferases in Golgi apparatus from rat liver. J. Biol. Chem. 249: Keller, G. A., T. J. Scallen, D. Clarke, P. A. Maker, S. K. Krisans, and S. J. Singer Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes. J. Cell Biol. 108: Kobayashi, T., and R. E. Pagano Lipid transport during mitosis. J. Biol. Chem. 264: Kok, J. W., S. Eskelinen, K. Hoekstra, and D. Hoekstra Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis. Proc.

17 VOL. 55, 1991 INTRACELLULAR LIPID MOVEMENT IN EUKARYOTES 559 Natl. Acad. Sci. USA 86: Koval, M., and R. E. Pagano Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J. Cell Biol. 108: Koval, M., and R. E. Pagano Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick type A fibroblasts. J. Cell Biol. 111: Kuchler, K., G. Daum, and F. Paltauf Subcellular and submitochondrial localization of phospholipid synthesizing enzymes in Saccharomyces cerevisiae. J. Bacteriol. 165: Kuge, O., M. NishUima, and Y. Akamatsu Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs. J. Biol. Chem. 261: Lambeth, J. D., X. X. Xu, and M. Glover Cholesterol sulfate inhibits adrenal mitochondrial cholesterol side chain cleavage at a site distinct from cytochrome P-450SCC. J. Biol. Chem. 262: Lange, Y., and H. J. G. Matthies Transfer of cholesterol from its site of synthesis to the plasma membrane. J. Biol. Chem. 259: Lange, Y., and T. Steck Cholesterol-rich intracellular membranes: a precursor to the plasma membrane. J. Biol. Chem. 260: Lange, Y., M. H. Swaisgood, B. V. Ramos, and T. L. Steck Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J. Biol. Chem. 264: Lipsky, N. G., and R. E. Pagano Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc. Natl. Acad. Sci. USA 80: Lipsky, N. G., and R. E. Pagano Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J. Cell Biol. 100: Liscum, L., and J. R. Faust Low density lipoprotein (LDL) mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts. J. Biol. Chem. 262: Liscum, L., and J. R. Faust The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-p-[2-(di-ethylamino) ethoxy]androst-5-en-17-one. J. Biol. Chem. 264: Liscum, L., R. M. Ruggiero, and J. R. Faust The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. J. Cell Biol. 108: Martin, D. C., and R. E. Pagano Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. J. Biol. Chem. 262: Metz, R. J., and N. S. Radin Purification and properties of a cerebroside transfer protein. J. Biol. Chem. 257: Morrot, G., P. Herve, A. Zachowski, P. Fellmann, and P. F. Devaux Aminophospholipid translocase of human erythrocytes: phospholipid substrate specificity and effect of cholesterol. Biochemistry 28: NishiJima, M., 0. Kuge, and Y. Akamatsu Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation. J. Biol. Chem. 261: Noland, B. J., R. E. Archalo, E. Hansbury, and T. J. Scallen Purification and properties of sterol carrier protein. J. Biol. Chem. 255: Novick, P., C. Field, and R. Schekman Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21: Op den Kamp, J. A. F Lipid assymetry in membranes. Annu. Rev. Biochem. 48: Ossendorp, B., G. P. H. Van Heusden, and K. W. A. Wirtz The amino acid sequence of rat liver nonspecific lipid transfer protein (sterol carrier protein 2) is present in a high molecular weight protein: evidence from cdna analysis. Biochem. Biophys. Res. Commun. 168: Pacuszka, T., R. 0. Duffard, R. N. Nishimura, R. 0. Brady, and P. H. Fishman Biosynthesis of bovine thyroid gangliosides. J. Biol. Chem. 253: Pagano, R. E The Golgi apparatus: insights from lipid biochemistry. Biochem. Soc. Trans. 18: Pagano, R. E., and K. J. Longmuir Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J. Biol. Chem. 260: Pagano, R. E., K. J. Longmuir, and 0. C. Martin Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts. J. Biol. Chem. 258: Pagano, R. E., and R. G. Sleight Defining lipid transport pathways in animal cells. Science 229: Pentchev, P. G., M. E. Comly, H. S. Kruth, J. D. Butler, M. T. Vanier, D. A. Wenger, and S. Patel Type C Niemann- Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts. J. Biol. Chem. 261: Pentchev, P. G., M. E. Comly, H. S. Kruth, T. Tokoro, J. Butler, J. Sokol, M. Filling-Katz, J. M. Quirk, D. C. Marshall, S. Patel, M. T. Vanier, and R. 0. Brady Group C Niemann-Pick disease: faulty regulation of low density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J. 1: Pentchev, P. G., M. E. Comly, H. S. Kruth, M. T. Vanier, D. A. Wenger, S. Patel, and R. 0. Brady A defect in cholesterol esterification in Niemann-Pick (type C) patients. Proc. Natl. Acad. Sci. USA 82: Phillips, M. C., W. J. Johnson, and G. Rothblat Mechanisms and consequences of cellular cholesterol exchange and transfer. Biochim. Biophys. Acta 906: Poznansky, M. J., and S. Czekanski Cholesterol movement between human skin fibroblasts and phosphatidylcholine vesicles. Biochim. Biophys. Acta 685: Reinhart, M. P., J. T. Billheimer, J. R. Faust, and J. L. Gaylor Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver. J. Biol. Chem. 262: Sandra, A., and R. E. Pagano Phospholipid asymmetry in LM cell plasma membrane derivatives: polar head group and acyl chain distributions. Biochemistry 17: Siegneuret, M., and P. F. Devaux ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes. Proc. Natl. Acad. Sci. USA 81: Siegrist, H. P., T. Burkhart, U. N. Wiesmann, N. N. Herschkowitz, and M. A. Spycher Ceramide-galactosyltransferase and cerebroside-sulfotransferase localization in Golgi membranes isolated by a continuous sucrose gradient of mouse brain microsomes. J. Neurochem. 33: Simbeni, R., F. Paltauf, and G. Daum Intramitochondrial transfer of phospholipids in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 265: Sleight, R. G., and M. N. Abanto Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines. J. Cell Sci. 93: Sleight, R. G., and R. E. Pagano Rapid appearance of newly synthesized phosphatidylethanolamine at the plasma membrane. J. Biol. Chem. 258: Sleight, R. G., and R. E. Pagano Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus. J. Cell Biol. 99:

18 560 VOELKER 85. Sleight, R. G., and R. E. Pagano Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells. J. Biol. Chem. 260: Tilley, L., S. Criber, B. Roelofsen, J. A. F. Op de Kamp, and L. L. M. van Deenen ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane. FEBS Lett. 194: Ting, A. E., and R. E. Pagano Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth. J. Biol. Chem. 265: Tsuneoka, M., A. Yamamoto, Y. Fujiki, and Y. Tashio Nonspecific lipid transfer protein (sterol carrier protein-2) is located in rat liver peroxisomes. J. Biochem. (Tokyo) 104: Urbani, L., and R. D. Simoni Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J. Biol. Chem. 265: Van Amerongen, A., J. B. Helms, T. P. Van der Krift, R. B. H. Schutgens, and K. W. A. Wirtz Purification of nonspecific lipid transfer protein (serol carrier protein 2) from human liver and its deficiency in livers from patients with cerebrohepato-renal (Zellweger) syndrome. Biochim. Biophys. Acta 919: Vance, D. E., and J. E. Vance Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes. J. Biol. Chem. 261: Vance, J. E Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265: Vance, J. E., E. J. Aaslman, and R. Szarka Brefeldin A does not inhibit the movement of phosphatidylethanolamine from its site of synthesis to the cell surface. J. Biol. Chem. 266: Vance, J. E., and D. E. Vance Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J. Biol. Chem. 263: Van der Krift, T. P., J. Leunissen, T. Teerlink, G. P. H. Van Heusden, A. J. Verklei, and K. W. A. Wirtz Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the nonspecific lipid transfer protein (sterol carrier protein 2). Biochim. Biophys. Acta 812: Van Golde, L. M. G., J. Raben, J. J. Batenburg, B. Fleischer, F. Zambrano, and S. Fleischer Biosynthesis of lipids in Golgi complex and other subcellular fractions from rat liver. Biochim. Biophys. Acta 360: Van Heusden, G. P. H., K. Bas, C. R. H. Raetz, and K. W. A. Wirtz Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2). J. Biol. Chem. 265: van Meer, G Plasma membrane cholesterol pools. Trends Biochem. Sci. 12: van Meer, G., E. H. K. Stelzer, R. W. Wjnaendts-van-Resandt, MICROBIOL. REV. and K. Simons Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J. Cell Biol. 105: Verklei, A. J., R. F. A. Zwaal, B. Roelofsen, P. Comfurius, D. Kastelln, and L. L. M. van Deenen The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etching electron microscopy. Biochim. Biophys. Acta 323: Voelker, D. R Phosphatidylserine functions as the major precursor of phosphatidylethanolamine in cultured BHK 21 cells. Proc. Natl. Acad. Sci. USA 81: Voelker, D. R Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells. J. Biol. Chem. 260: Voelker, D. R Lipid assembly into cell membranes, p In D. E. Vance and J. E. Vance (ed.), Biochemistry of lipids and membranes. The Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif Voelker, D. R Phosphatidylserine translocation to the mitochondrion is an ATP dependent process in permeabilized animal cells. Proc. Natl. Acad. Sci. USA 86: Voelker, D. R Reconstitution of phosphatidylserine import into rat liver mitochondria. J. Biol. Chem. 264: Voelker, D. R Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells. J. Biol. Chem. 265: Voelker, D. R Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-Kl cells. J. Biol. Chem. 266: Voelker, D. R., and J. L. Frazier Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and inhibiting defective phosphatidylserine synthase activity. J. Biol. Chem. 261: Wattenberg, B Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system. J. Cell Biol. 111: Wirtz, K. W. A., and T. W. J. Gadelia Properties and modes of action of specific and nonspecific phospholipid transfer proteins. Experientia 46: Wirtz, K. W. A., and D. B. Zilversmit Exchange of phospholipids between liver mitochondria and microsomes in vitro. J. Biol. Chem. 243: Yaffe, M. P., and E. P. Kennedy Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells. Biochemistry 22: Zachowski, A., E. Favre, S. Cribier, P. Hervd, and P. F. Devaux Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme. Biochemistry 25: Zborowski, J., A. Dygas, and L. Wojtczak Phosphatidylserine decarboxylase is located on the external side of the inner mitochondrial membrane. FEBS Lett. 157: