ALL EUKARYOTIC cells secrete proteins. Higher eukaryotic
|
|
- Marshall Fletcher
- 5 years ago
- Views:
Transcription
1 X/98/$03.00/0 Endocrine Reviews 19(2): Copyright 1998 by The Endocrine Society Printed in U.S.A. Endocrinopathies in the Family of Endoplasmic Reticulum (ER) Storage Diseases: Disorders of Protein Trafficking and the Role of ER Molecular Chaperones* PAUL S. KIM AND PETER ARVAN Division of Endocrinology (P.S.K.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Division of Endocrinology (P.A.) and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York I. Introduction A. Overview B. Protein folding in the ER C. Supervised folding: the concept of molecular chaperones and folding catalysts D. Co- and posttranslational modifications are factors that can influence folding II. ER Molecular Chaperones, Folding Catalysts, and Molecular Escorts A. Binding protein (BiP) B. GRP94 C. Calnexin and calreticulin D. Disulfide isomerase and prolyl isomerase: families of folding catalysts E. ERp72 and ER60 F. HSP47 G. Molecular escorts: pro-peptides, transport subunits, receptor-associated protein (RAP), and 7B2 III. Models of ER to Golgi Traffic Influence Models of Quality Control A. Escape from ER retention as one hypothesis to explain anterograde protein traffic from the ER B. Cargo receptors as another hypothesis to explain anterograde protein traffic from the ER C. What provides quality control of ER export? D. ER-associated degradation IV. Endocrinopathies as Models of Defective Protein Export A. Congenital hypothyroid goiter with thyroglobulin deficiency B. Familial neurohypophyseal diabetes insipidus C. Osteogenesis imperfecta and disorders of procollagen biosynthesis D. ERSDs affecting lipoprotein metabolism E. Other selected nonendocrine and endocrine ERSDs V. Summary: A Proposed Classification of ERSDs Address correspondence to: Peter Arvan, M.D., Ph.D., Division of Endocrinology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York *This work was supported by NIH Grants DK to (P.A.) and DK (to P.S.K.), as well as support from Knoll Pharmaceuticals. I. Introduction A. Overview ALL EUKARYOTIC cells secrete proteins. Higher eukaryotic tissues, in general, and many endocrine glands, in particular, are differentiated to release abundant quantities of specialized proteins. Most of the proteins released from cells are carried to the plasma membrane via the biosynthetic transport pathway. The entire pathway is comprised of specific transport vesicles that shuttle their cargo through a series of intracellular way-stations (1): at each successive station, specific sorting decisions can be made on the basis of transport signals (2, 3) and retention signals (4 6). Exportable proteins enter at the endoplasmic reticulum (ER), 1 the first membrane-bounded compartment of this pathway (7, 8). Functions of the ER include the synthesis, initial modification, and export of polypeptides destined for secretion or to be located at the plasma membrane. One of the most important jobs of the ER is to provide an environment to facilitate the proper folding and assembly of newly synthesized exportable proteins. In addition, the ER contains mechanisms to monitor the fidelity of these early biosynthetic events in the protein export pathway. This has been called ER quality control (9), which involves machinery 1 The following abbreviations are used: ER, endoplasmic reticulum; ERSD, ER storage disease; HSP, heat shock protein; GRP, glucoseregulated protein; BiP, immunoglobulin heavy chain-binding protein; SREBP, sterol-regulatory element-binding protein; CHOP, C/EBP homologous protein; GADD153, growth arrest and damage-inducible gene 153; GPI, glycosylphosphatidylinositol; UGGT, UDP-glucose:glycoprotein-glucosyltransferase; GlcNac, N-acetyl glucosamine; Man, mannose; PDI, protein disulfide isomerase; C-X-X-C, cysteine-x-x-cysteine; PPI, peptidylprolyl isomerase; FKBP, FK506 binding protein; Erp72, endoplasmic reticulum protein 72; ER60, endoplasmic reticulum protein 60; RAP, receptor-associated protein; LDL, low-density lipoprotein; LRP, low-density lipoprotein-receptor related protein; PC, prohormone convertase; KDEL, lysine-aspartate-glutamate-leucine; COP, coat protein complex; ERGIC53, a protein residing in the compartment intermediate between ER and Golgi; MHC, major histocompatability complex; ERAD, ER-associated degradation; Tg, thyroglobulin; FDI, familial diabetes insipidus; DI, diabetes insipidus; AVP, vasopressin; NP, neurophysin; OI, osteogenesis imperfecta; LDL-R, low-density lipoprotein receptor; MTP, microsomal triglyceride transfer protein; LPL, lipoprotein lipase; AAT, -1-antitrypsin; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator. 173
2 174 KIM AND ARVAN Vol. 19, No. 2 designed to try to prevent premature export of incompletely or improperly folded proteins from the ER, as well as machinery intended to initiate the removal of misfolded, incompetent proteins (10). These features of the ER have evolved to reduce potential harm posed by exportable proteins that are prone to aggregation and malfunction. Thus, ER quality control machinery is designed to differentiate normal and abnormal forms of a wide variety of exportable proteins, presumably by recognizing structural signals that are enriched in misfolded and incompletely folded molecules. Recent years have witnessed the identification of numerous inborn errors of metabolism that affect secretory or plasma membrane proteins. In many instances, mutations causing minor changes in protein primary structure lead to intracellular retention of the affected proteins, suggesting that proper conformation is critical for protein transport as well as biological activity. Nowadays, scientists are increasingly combining molecular and biochemical analyses in the hopes of identifying precisely how mutations produce folding defects that lead to abnormal protein trafficking. In this report, we review defective protein export as the cause of a variety of endocrinopathies that fall into the category of Endoplasmic Reticulum Storage Diseases (ERSDs) (11). These disorders include certain forms of congenital hypothyroid goiter, osteogenesis imperfecta, diabetes insipidus, familial hypercholesterolemia, and others. In each case, the disease leads to accumulation in the ER of a critically important protein that is unable to reach its target site and therefore is unable to perform its physiologically intended function. Morphological studies of cells affected by ERSDs routinely reveal expansion and dilation of the ER compartment, which may in part be due to accumulation of misfolded exportable proteins. Moreover, the compensatory response in such cells also frequently includes a selective induction of the synthesis (and supranormal accumulation) of several ER resident proteins that are thought to participate in ER quality control. Most of these proteins are considered to be molecular chaperones, a subtopic that is considered further in Section II. This review will provide an endocrinologist s perspective of protein folding in the ER. From this vantage point, we consider how subtle mutations in the coding sequences of polypeptide hormone precursors and other exportable proteins, in conjunction with ER quality control, can lead to defective protein trafficking, causing a disease phenotype. Finally, we review specific representative endocrinopathies in greater detail, as a means to highlight the underlying similarities and differences in phenotypes and modes of transmission of ERSDs, with an eye toward identifying future directions of endocrine investigation. B. Protein folding in the ER Until recently, the principle of protein biogenesis relied entirely on the hypothesis that each peptide chain can selfassemble into a stable, low free-energy conformation, based solely on information contained within the primary structure (12, 13). The idea that few additional factors were required for proper folding was supported by studies of the renaturation of small polypeptides in dilute solution at reduced temperature. However, folding of proteins in living cells (14), especially within the ER compartment, occurs under highly restrictive conditions unique to this microenvironment (15, 16). For one thing, the protein concentration in the lumen of the ER may be as high as 100 mg/ml (17). Second, secretory proteins are translocated into the ER as they are being translated; thus, the NH 2 termini of secretory proteins routinely begin to fold in the ER before the COOH termini have even been synthesized (18, 19). Third, the ER is the site for de novo addition of N-linked core oligosaccharides, as well as initial carbohydrate processing that may exert both direct and indirect effects on glycoprotein folding (see Section I.D). Fourth, the composition of ions and small molecules in the intraluminal environment of the ER is highly regulated: this includes levels of calcium (20) and ATP (21). Finally, in mammalian cells, the ER is a more oxidizing environment than the soluble cytoplasm, owing to the transport of oxidized glutathione (22), which fosters the formation of disulfide bridges (23). All of these conditions, but particularly the oxidizing environment and extraordinary concentration of nascent (unfolded) polypeptides, lead to an increased possibility for improper intra- and intermolecular associations. In spite of these obstacles, a high fraction of newly synthesized secretory and plasma membrane proteins are successfully folded and exported from the ER. Indeed, while a fraction of the initial cohort of newly made exportable proteins may enter novel misfolded states, for endogenous proteins in general, most of the cohort follows a statisticallymost-probable folding pathway, proceeding through a predictable series of conformational intermediates en route to the native state (24 28). It is believed that this process has evolved via cotranslational domain-dependent folding (19) in conjunction with the actions of compartment-specific molecular chaperones. C. Supervised folding: the concept of molecular chaperones and folding catalysts It is believed that the overall speed and efficiency of exportable protein folding is enhanced through a combination of interactions with another group of proteins resident to the ER. This group includes members of highly conserved families of molecular chaperones, as well as others to be described, some of which are viewed primarily as folding catalysts. Found in every living cell, molecular chaperones were originally defined as families of proteins that assist in the self-assembly of other chains but are generally not part of the final functional unit (29). Thus, by definition, classical molecular chaperones interact only transiently with their substrate proteins (for further definition, see Section II and Fig. 1, below). Of the ER molecular chaperones, several are known to be members of the larger family of heat shock proteins (HSPs), conserved even to prokaryotes (which lack ER and other cytoplasmic organelles). Heat shock is only one of several different types of stress that can cause protein denaturation, and HSP60, HSP70, and HSP90 classes of heat shock proteins, so named for their approximate molecular weights, are known to play crucial compensatory roles that allow cell survival in the face of stress, by limiting and po-
3 April, 1998 ENDOCRINE ERSDs 175 FIG. 1. Categories of ER helper proteins described in the text. The relative abundance of many ER resident proteins varies considerably with cell type and is stimulated under conditions of cellular stress (see text), but many of these molecules are very abundant, perhaps on the order of 10 mg/ml, even under resting conditions (92, 457). This schematic figure divides helper proteins in the ER into four general categories that are defined in the text at the beginning of Section II. These groupings are not complete lists, e.g., the protein-specific ER chaperones do not include BAP31, an ER membrane protein that interacts selectively with certain members of the cellubrevin/synaptobrevin family (459). Some proteins listed are not discussed individually in the text. Under the enzymes that regulate folding are included molecules that may have both direct and indirect effects on protein conformation, e.g., sugar processing in the ER may directly influence folding as well as by glycoprotein association with calnexin and calreticulin (see Section II.C). Other molecules listed fall into gray areas with respect to category. For example, PDI has been described as both a foldase and a classic ER chaperone (see Section II.D). Likewise, it is possible that ERp72 and/or ER60, listed here as classic chaperones, might exhibit PDI-like catalytic activity in vivo. Further, prolyl hydroxylase and glutamyl carboxylase, although widely expressed among different tissues, might be considered as protein-specific. Moreover, classic ER chaperones may also have the potential to promote the folding and export of some secretory proteins and promote the degradation of others (see Section III). Finally, a new category of proteins are listed here here under the name of molecular escorts (see Section II.G). In some cases, a secretory propeptide may be important for nascent chain folding and may therefore be considered as a protein-specific chaperone, but the pro-region frequently escorts the rest of the polypeptide through the secretory pathway, warranting inclusion in both categories. RAP is similarly considered in these two categories. The common -subunit of glycoprotein hormones is considered here as a molecular escort, especially in the case of FSH or TSH (246); however, and -subunits of glycoprotein hormones remain associated even after secretion, where the heterodimeric structure may be important for biological activity (see Section II.G). tentially reversing aggregation of misfolded proteins (30). In the cells of higher eukaryotes, each intracellular compartment, including the ER, has its own subset of HSPs and other chaperones (except for the absence of an identifiable HSP60 class member in the ER). In the HSP70 class, all members share remarkable homology in their substrate -binding regions and the same is true of the HSP90 class. Thus members within a given class tend to differ mostly within the region of the chaperone that specifies targeting to a particular subcellular compartment. However, certain metabolic insults may tend to trigger a chaperone stress response limited more or less selectively to a specific compartment (31 33). Indeed, glucose deprivation is a relatively selective stimulant of the ER stress response; thus, certain ER chaperones also go under the name of GRPs, for glucose-regulated proteins (34). Generally speaking, HSPs are abundantly expressed under normal conditions but their synthesis is further induced under stress conditions. The regulation of ER chaperone levels is a complex process that has been reviewed elsewhere (35), although recent papers have shed increasing light on the signaling pathway responsible for induction of chaperone synthesis in response to the presence of accumulating unfolded secretory proteins now known as the unfolded protein response (36). It had been suspected for some time that a signaling cascade for increased synthesis of the ER-HSP70 family member (known as BiP) begins with an increase in the ratio of bound/unbound chaperone (37). A reduction in the free level of chaperones [as a consequence of increased association with binding sites on available incompletely folded proteins (38, 39)] leads to the activation of one (40 42) or more (43) possible protein kinases, triggering a signal that induces further synthesis of BiP and other ER chaperones (31, 44) through what is thought to be a predominantly transcriptional mechanism (45). The kinase IRE-1, which transmits its signal across the ER membrane, is a type 1 membrane protein whose N terminus (in the ER lumen) has no homology to other proteins, while its C terminus (in the cytosol) contains a predicted serine kinase. The activity of IRE-1 transcriptionally regulates the stability of a specific transcription factor, HAC1 (46, 47). Other factors implicated in the unfolded protein response may include sterol-regulatory element-binding proteins (48), induced by sterol depletion (36), as well as CHOP (also known as GADD153) which is a member of the C/EBP family of transcriptional factors that can be markedly induced as a consequence of certain forms of ER stress (45). The accumulation of proteins in the ER membrane may trigger a second distinct signal transduction pathway, recently termed the ER-overload response (36). In this case, increasing presence of either misfolded or nascent membrane proteins is capable of causing the activation of another transcriptional factor, nuclear factor- B, which regulates a different cascade of gene expression (49). Some forms of cellular stress can trigger both unfolded protein and ER-overload responses, while others are selective for only one signaling pathway. In addition to these transcriptional mechanisms, cells appear to be able to posttranslationally regulate ER chaperone activity, to a variable extent, via ADP ribosylation (50), phosphorylation (51 55), as well as formation of oligomeric chaperone complexes (56).
4 176 KIM AND ARVAN Vol. 19, No. 2 To the extent that HSP classes of ER chaperones monitor protein folding, the transcriptional regulation described above is a mechanism designed to ensure that adequate availability of these supervisory molecules is maintained at all times. The importance of available free chaperones is underscored by the recent understanding that even after dissociation from a given chaperone, incompletely folded substrate proteins routinely rebind to the same or another copy of that chaperone, contributing to an increase in the bound chaperone fraction. Although the structural basis for the binding of different chaperones appears to vary, the property of cyclic association-dissociation is a common feature ranging from the bacterial chaperonins (57) to ER chaperones that are primarily involved in recognition of polypeptide (58) or carbohydrate moieties (9). Importantly, the fact that molecular chaperones act on substrate proteins does not violate the principle of self-assembly. As a rule, classic chaperones interact with many different substrates without conferring steric information to influence the final folded structure. However, by interacting with nascent chains, chaperones prevent (and may even reverse) undesirable protein-protein interactions; this increases the chances that newly made proteins will have the opportunity to achieve their native structure. Because ER chaperone associations are based on recognition of features enriched in incompletely folded versions of exportable proteins, associations of ER chaperones are usually (but not always) at their highest levels immediately upon nascent chain translocation into the ER, and terminated before export of the substrate protein from this compartment. Not every interaction with individual ER resident proteins will enhance folding speed or even folding efficiency (see Section III), although some available data tend to suggest enhancement of efficiency at the possible expense of delayed protein folding (59). Promotion of productive folding is likely to be the net effect of interactions with both chaperones and folding catalysts. However, the extent to which roles played by the binding of individual chaperones are overlapping vs. unique (60), and how the different chaperones may cooperate in the folding process for a wide variety of proteins (14), remains largely unknown. D. Co- and posttranslational modifications are factors that can influence folding Although the flow of genetic information ends when the primary structure has been synthesized, co- and posttranslational modifications, under the influence of local environmental factors (discussed in Section I.B), can also affect the folding outcome for many exportable proteins. Of course, many important modification steps (e.g., terminal glycosylation, sulfation, phosphorylation, and dibasic proteolytic cleavage events) take place as proteins are transported through the Golgi complex, which can significantly alter protein destination and biological function. However, with the exception of proteolytic cleavage, Golgi processing activities generally have fewer effects on underlying protein conformation than the processing activities of the ER, which are the subject of this section. One of the most important ER modifications is the proteolytic cleavage of the predominantly hydrophobic 20 amino acid signal sequence (61) by the signal peptidase complex; the signal peptide is degraded after it has served to target the nascent chain into the ER translocation channel (62). Indeed, failure to remove the signal peptide generally results in severe, irreversible misfolding of secretory proteins (63). Also, as the nascent chain enters the ER lumen, the rapid collapse of its hydrophobic domains is accompanied by the ordered formation of intramolecular disulfide bonds (26, 64), which stabilize secondary and tertiary structure and can be critical for maintaining a biologically active conformation (65). Indeed, most of the cysteine residues of exportable proteins eventually form disulfides (23), while similar covalent bonds are not observed in cytosolic proteins or in the cytosolic domains of transmembrane proteins. Within the oxidizing ER environment, reactive thiols also can form mispaired disulfide bonds. Subsequent reshuffling and correction of aberrant disulfide bonds (see Section II.D) may represent one of the rate-limiting steps in protein folding (23). Studies of exportable proteins mutated to lack specific cysteine residues have provided additional evidence for the importance of correct disulfide bond formation. Similarly, treatment of live cells with dithiothreitol or other membrane-permeant reductants results in unfolding of many newly made proteins; upon removal of dithiothreitol, reduced proteins begin to properly refold, reoxidize, and ultimately leave the ER, albeit at a slower rate (66 71). Intermolecular disulfide bonds may also be important in the formation and maintenance of quaternary structure (28, 72). One of the next most important ER modifications is the addition of N-linked carbohydrates to glycoproteins (73). A large preassembled, oligosaccharide containing two N-acetylglucosamines, nine mannoses, and three terminal glucoses (74) is transferred cotranslationally from a dolichollinked intermediate to an asparagine residue (of the consensus sequence, Asn-X-Ser/Thr), as the nascent polypeptide emerges through the translocation channel in the ER membrane (Fig. 2). ER membrane proteins known as ribophorins (75), as part of a protein complex encoded by at least seven genes (76), assist in the catalysis of this initial glycosylation reaction. Further ER carbohydrate modifications then occur through the actions of glucosidases and other processing enzymes (Fig. 2, discussed in Section II.C). Although not found on all exported proteins, carbohydrate moieties often assist in the folding, stability, and solubility of nascent exportable polypeptides (77), and in some cases glycosylation is required for the folding of subunits that occurs before oligomeric assembly (78). Thus, it is not surprising that inhibition of N-linked glycosylation frequently leads to misfolding and aggregation of nascent chains. Once fully folded, however, removal of sugar groups generally has little impact on protein solubility and conformation. Quaternary structural maturation is another important modification, which can range from simple homodimers to larger hetero-oligomeric complexes that are associated either covalently or noncovalently (79). Although there are exceptions (79a), proper oligomeric assembly is frequently
5 April, 1998 ENDOCRINE ERSDs 177 FIG. 2. N-Linked oligosaccharide processing of exportable glycoproteins in the ER. N-Linked carbohydrates may be added to Asn-residues of exportable proteins containing the consensus acceptor sequence Asn-X-Ser/Thr. The N-linked carbohydrate is added en bloc via a dolicholphosphate intermediate. The two proximal N-acetyl glucosamine residues are represented as dark, filled ovals. The triantennary Man 9 structure is shown as ovals containing the letter M. The terminal glucose residues are indicated as small boxes containing the letter G. Shortly after oligosaccharide addition, monosaccharide removal begins, first with glucosidase 1 removing the outermost glucose residue and glucosidase 2 removing the next outermost residue. These glucosidase reactions can be pharmacologically inhibited with deoxynorjirimicin or castanospermine (see Section III.C). It is the remaining monoglucosylated carbohydrate form that has been shown to interact with calnexin and calreticulin (see text). A single terminal glucose may be restored onto Man 9 (or even Man 8 or Man 7 ) by the action of UGGT, which is thought to act in a cyclical, repetitive fashion on deglucosylated glycoproteins that are sensed by the enzyme to be misfolded. Such reglucosylation stimulates reassociation with calnexin and calreticulin (see text). Mannosidase action may begin before, but occurs largely after, the glucose cycling is completed; importantly, trimming below the Man 7 stage may eliminate the oligosaccharide from further suitability as a substrate for the UGGT enzyme. required for ER to Golgi transport of secretory and plasma membrane glycoproteins, whereas unassembled monomers are often retained in the ER until the protein is degraded or an assembly partner becomes available (72). Other covalent modifications that can also have profound effects on protein folding may be limited to certain subgroups of exported proteins. For example, cotranslational hydroxylation of proline within the tripeptide Gly- Pro-Pro repeats of collagen, catalyzed by an ER enzyme known as prolyl-4-hydroxylase, is essential for triple-helix formation and stability (80, 81). Hydroxylation of lysine side chains also plays an important role in collagen stability (82, 83). Similarly, carboxylation of glutamyl residues by a vitamin K-dependent mechanism plays an important structural role in the stability of a number of bloodclotting factors and calcium-binding proteins (84, 85). Moreover, covalent attachment of a glycosylphosphatidylinositol anchor near the carboxy termini of a certain subset of exportable proteins is associated with the proteolytic cleavage of the C-terminal 20 amino acids (86, 87); failure to remove these residues can lead to protein aggregation and transport incompetence (88). Other roles for glycosylphosphatidylinositol anchors in protein folding and ER export per se currently remain unknown. II. ER Molecular Chaperones, Folding Catalysts, and Molecular Escorts For antibodies to identify antigenic proteins, an entire system exists for gene rearrangement to produce Ig variable regions intended to serve as high-affinity peptide-interaction sites for diverse recognition of foreign products. The complexity of this situation can be loosely analogized to the ER, where a wide range of small peptides are exposed, in the context of nascent polypeptide folding, that are normally not exposed in the respective native structures. Although generally hydrophobic, there is undoubtedly considerable diversity in the primary structures exposed in unfolded patches of nascent exportable polypeptides. For this reason in eukaryotic cells, a system has evolved to minimize exposure of these unfolded patches and thereby decrease the risk of promoting improper intrachain and interchain peptide interactions. However, instead of being based on gene rearrangement, the system of recognition of unfolded proteins in the ER involves a finite series of genes producing proteins whose peptide-interaction sites tend to be more promiscuous than those of antibodies. Concomitant with this promiscuity is a tendency toward lower affinity interaction with any particular polypeptide. By differences in peptide interaction specificities of different chaperones, and promiscuity of in-
6 178 KIM AND ARVAN Vol. 19, No. 2 teraction of each chaperone, as well as the sheer concentration of chaperones in the ER, dynamic interactions with a wide range of substrates are produced. This idea underscores the basic mechanism of ER quality control, to be discussed further (Section II.C). For the present, we wish to describe properties of a selected subset of known 1) ER chaperones, 2) folding catalysts (Section II.A F), and 3) molecular escorts (Section II.G) in the protein export pathway (Fig. 1). In conjunction with the schema in Fig. 1, we define these molecules as follows: 1) ER chaperones are simply viewed as binding proteins whose association with exportable protein substrates is regulated by the concentrations of the two components and their binding affinity in a bimolecular interaction. 2) Folding catalysts are true enzymes, which also physically interact with substrate proteins, but in so doing they lower the activation energy required for a discrete conformational change within an exportable protein. In the secretory pathway, chaperones and folding catalysts tend to reside predominantly in the ER, where they serve their primary biological functions, while 3) transport subunits and molecular escorts routinely accompany their substrate proteins out of the ER, and persistent interaction may even be required for ER exit. Note that we have not attempted to review each individual molecule listed in Fig. 1. A. Binding protein (BiP) BiP (also known as GRP78), the most studied ER molecular chaperone, is a member of the HSP70 class (89 91), a major calcium-binding protein in the ER lumen (92), and an essential gene product that even the simplest eukaryotes cannot live without (93, 94). Like other members of the HSP70 class, BiP possesses a peptide-binding groove lined with hydrophobic side chains, which is thought to interact optimally with hepta- or octapeptides enriched in aromatic/ hydrophobic residues in alternating positions a feature common to many naturally occurring peptide chains (95 97). In properly folded globular proteins, such features are normally buried in the hydrophobic core or are used at the interface between subunits for protein oligomerization. This helps to account for the observation that although BiP interacts with a remarkably wide range of exportable proteins, it tends to associate most strongly with unfolded, unassembled, or aberrant polypeptides (25, ). In addition to the role of BiP in posttranslational folding, this chaperone has been proposed to be involved in the translocation of nascent polypeptides across the ER membrane ( ). Further, BiP plays an indirect but key role in the fusion of nuclear membranes during fertilization between haploid yeast cells (112, 113). Although the physiological relevance of these functions for mammalian cells in vivo remains to be determined (114), it is presumed from these studies that luminal BiP binding serves to stabilize nascent polypeptide chains as well as the luminal domains of certain endogenous ER membrane proteins. Misfolded exportable proteins, such as those produced by mutation or abnormal glycosylation, have been found to interact with BiP for periods long after their synthesis (e.g., Refs ). Such prolonged interaction occurs via persistent reassociation of the chaperone (58) to BiP-binding sites that cannot be properly buried within the hydrophobic core of misfolded polypeptides. Because each round of BiP dissociation is coupled to a round of ATP hydrolysis (56, 118), ATP consumption in the ER of cells that have accumulated misfolded polypeptides is expected to increase. Further, misfolding of exportable proteins can be found in cells depleted of ATP (66, 119); this observation has been used to suggest that cyclical binding of BiP and other ATP-dependent chaperones facilitates protein maturation (15). However, it must be pointed out that because ATP hydrolysis is required for polypeptide release from BiP (120), the depletion of ATP or the use of BiP mutants that cannot hydrolyze ATP ( ) produces defective chaperone dissociation that is likely to inhibit the maturation and ER egress of exportable proteins (see Section III). Thus, the results of experiments using these approaches cannot and do not independently establish that BiP binding normally promotes folding or export from the ER under physiological conditions, although such a conclusion has been suggested based on in vivo protein refolding after heat shock (125). En route to normal folding, assembly, and exit from the ER, BiP binding to exportable proteins is typically observed only transiently (25, 126, 127). Importantly, existence of such transient interaction also cannot distinguish models in which BiP binding is thought to promote folding and export from those where it is thought to delay folding and export (see Section III). In addition, because individual BiP-binding sites are represented by relatively small stretches of primary structure, incompletely folded versions of large polypeptides may expose multiple potential BiP-binding sites during protein folding, simultaneously or in series (71). For example, at a moment in time in the steady state, the average stoichiometry of association between BiP and nascent thyroglobulin (a 330-kDa monomer) has been estimated at 10:1 in the thyroid ER (25). Potentially, the earliest folding intermediates of nascent secretory proteins may bind even more than the average number of BiP molecules, while later, more folded forms are likely to associate with progressively fewer BiP molecules. As described in an earlier section, BiP levels are transcriptionally regulated by the unfolded protein response. Importantly, ordinary physiological dynamics of the production of exportable proteins is sufficient to regulate the synthesis of BiP and other ER chaperones ( ). Lastly, coupled with the recent identification of 14 hsp70 family members in the yeast genome (59), a second, novel hsp70 member in the ER, LHS-1, has been described, which is a nonessential gene that exhibits partial BiP-like function (131). However, it is possible that in mammalian cells the function of the yeast LHS-1 is subserved by its non-hsp70 homolog, GRP170 (132). B. GRP94 GRP94 (also known as endoplasmin), the product of a single gene (133), is a member of the HSP90 class (134) and is also a major ER luminal calcium-binding protein (92, 135) that is transcriptionally coregulated with BiP under most conditions (34, 35). Considerably less is known about the peptide-binding specificity of GRP94, but by analogy to other
7 April, 1998 ENDOCRINE ERSDs 179 members of the HSP90 class, GRP94 is likely to act in a cooperative way (136, 137) in associating with nascent polypeptides that have exposed unfolded patches. For instance, GRP94 might be one of the luminal proteins whose binding enhances completion of nascent chain translocation into the ER lumen (110). Further, BiP and GRP94 are found in ternary complexes in which they are simultaneously involved in direct interactions with exportable proteins (138, 139), although their precise association-dissociation kinetics may not be identical (140). Like BiP, GRP94 can also be found to interact with misfolded exportable proteins for prolonged periods after synthesis (117, 138, ), as well as for transient periods with normal proteins maturing in the export pathway (140, 143). Individual polypeptides may expose more than one potential GRP94 binding site during folding, either simultaneously or in series (140). Sequence analysis has revealed two potential ATP-binding sites in GRP94 (53), which exhibits increased binding to polypeptide substrates under conditions of ATP depletion (33, 144). This and other observations led to the conclusion that GRP94 binds ATP and exhibits weak ATP hydrolytic activity (21, 145). However, a recent study has demonstrated that GRP94 interactions with peptides are nucleotide-independent, and that ATP binding and hydrolysis are not inherent properties of this chaperone (146). Such an interpretation is of particular interest because an adenine nucleotide-binding site has recently been unambiguously identified in the cytoplasmic homolog, hsp90 (147). Thus, although the molecular mechanism remains poorly understood, it is presumed that GRP94, like BiP, can undergo cycles of unbinding and rebinding to peptide substrates (15). It should be noted that thus far (148), such binding of GRP94 has not been demonstrated to result in enhancement of folding or export of proteins from the ER (139). As noted above, GRP94 levels are transcriptionally coregulated with those of BiP as part of the unfolded protein response (55, 130, 149). However, there are notable examples where disproportionate changes in one chaperone over the other is observed (150), indicating subtleties in transcriptional and/or translational regulation that are not well understood. C. Calnexin and calreticulin Calnexin, the only major molecular chaperone of the ER that is an integral membrane protein (151), is a single-spanning, calcium-binding protein of the ER membrane (152, 153). The CNX1 gene, a homolog that is believed to encode a form of calnexin from the yeast Saccharomyces pombe, is an essential gene whose critical function is contained within its ER luminal domain (154, 155). The binding/recognition function of mammalian calnexin has been an area of intense interest; almost immediately it was realized that treatment of cells with tunicamycin, a condition that causes severe misfolding of glycoproteins (and generally causes their increased binding to BiP), interferes with the binding of many such glycoproteins to calnexin (153). Nevertheless, calnexin does indeed bind to misfolded, mutant proteins and to folding intermediates of proteins en route to export ( ). The carbohydrate dependence of calnexin binding has led to the proposal that calnexin is a lectin (159, 160) serving as part of a chaperone apparatus that includes two independent enzyme activities: glucosidase II and UDP-glucose:glycoprotein-glucosyltransferase (UGGT) (161). This proposal is based upon knowledge of the processing pathway for N-linked carbohydrates in the ER (Fig. 2). A 14-saccharide unit is initially added en bloc to N-linked consensus acceptor sites in glycoproteins. This oligosaccharide includes two N-acetyl glucosamine (GlcNac) residues anchored in series to an asparagine in the exportable polypeptide (Fig. 2). Attached to this disaccharide is a triantennary structure comprised of nine mannoses (called Man 9 ). At one antenna of the Man 9 are a string of three terminal glucose residues (74). Normally, the three terminal glucoses are removed from N-linked oligosaccharides before glycoprotein exit from the ER, by the sequential action of glucosidase I (which removes the outermost glucose) and glucosidase II (which sequentially removes the remaining two glucose residues) (162). However, a single terminal glucose residue is restored onto Man 9 if the UGGT enzyme senses the glycoprotein to be unfolded (163, 164). This sensing involves interaction of the UGGT enzyme both with exposed hydrophobic residues as well as an exposed GlcNac residue at the base of the peptide-bound oligosaccharide (165). Calnexin in turn has been proposed to preferentially bind to the Glucose 1 -Man 9 -GlcNac 2 form of exportable glycoproteins in the ER (77, 166). All carbohydrate-binding activity of calnexin resides in its luminal domain (167). Recently, association of the calnexin luminal domain with monoglucosylated RNase B in vitro was shown to be very dynamic, suggesting that calnexin undergoes rapid on/off binding to monoglucosylated glycoproteins (167). Glucosidase II removal of terminal glucoses is not likely to occur while glycoproteins are bound to calnexin (167); however, during the period when glycoproteins have been released from calnexin, glucosidase II can remove the terminal glucose such that rebinding to calnexin cannot occur. In this view, cycles of rebinding to calnexin (168, 169) are triggered solely by the action of the UGGT enzyme. In contrast with the view of calnexin acting solely as a lectin in the ER lumen, others have suggested chaperone function involving the transmembrane, nonlectin portion of calnexin (170, 171) or chaperone binding to transmembrane domains of exportable proteins (172). In addition it has been shown that calnexin- substrate complexes are not dissociated even when the oligosaccharide is cleaved from the polypeptide upon endoglycosidase digestion (166, 173, 174), suggesting that some interactions with calnexin might occur in a glycan-independent manner (175). Further, certain unglycosylated proteins have also been shown (71, 176, 177) to associate with calnexin. Calnexin binding to unglycosylated proteins occurs commonly with protein aggregates that have been suggested to be separate from the productive maturation pathway (178). However, there is reason to believe that protein aggregates may indeed participate in productive maturation (25, 179); moreover, such aggregates bound to calnexin are reversible in vivo, leading to successful export from the ER (71). Thus, while the capability of calnexin to interact with monoglucosylated glycoproteins is unequivo-
8 180 KIM AND ARVAN Vol. 19, No. 2 cally established (167), whether this represents the complete story of its role in physiological protein maturation continues to be debated (180, 181). Calnexin is thought to be in close proximity to nascent chains upon their entry into the ER lumen; thus, it is not surprising that calnexin is hypothesized to play a cotranslational as well as posttranslational role in the folding of exportable proteins (71, 182). During the cotranslational period, calnexin binding may act to shield reactive free thiols from forming incorrect disulfide bonds, although this improvement in folding efficiency may actually slow down kinetic progression through the folding pathway (168, 183). Of course, these features are not discordant with observations that posttranslational rebinding of calnexin to monoglucosylated side chains (184) is also correlated with productive folding of exportable glycoproteins (169). Calreticulin (181, 185), a homolog of calnexin, is another major calcium binding protein (92, 135), the topology of which is entirely luminal. Although its function as part of an ER chaperone apparatus has been studied far less than that of calnexin (186), it shares with calnexin the capability of recognizing monoglucosylated glycoproteins, with partially overlapping specificity (187, 188). Calreticulin and calnexin both go through cycles of binding and release, indicating roles in monitoring and assisting protein folding (168, 169). Importantly, however, calreticulin is a full participant in the transcriptionally regulated, ER unfolded protein response (189), a feature noted to be absent for calnexin (151). Calreticulin also plays a role in intracellular calcium signaling (190, 191), in transcriptional regulation of steroid-sensitive gene expression (192), and in a number of other important cellular functions (reviewed separately in Ref. 185). Calmegin is the newest member of the calnexin family, found only in the testis, where it is thought to perform a function in the folding of proteins that are necessary for the ability of sperm to adhere to and fertilize eggs (193). D. Disulfide isomerase and prolyl isomerase: families of folding catalysts Protein disulfide isomerase (PDI), another major component of the ER lumen, is a true foldase, in that it catalyzes thiol-disulfide interchange with a broad substrate specificity, and it shows strong homology with bacterial thioredoxin (23, 194, 195). The regulation of PDI synthesis overlaps only partially with those of BiP, GRP94, calreticulin, and other ER residents that exhibit the unfolded protein response (189, 196). Instead, PDI expression seems to be proportional to the flux of nascent chains into the ER [i.e., especially high in secretory tissues ( )], and its activity may be further regulated posttranslationally (200). Recent reports indicate that PDI may undergo dimerization, autophosphorylation, and ATP hydrolysis that is stimulated in the presence of denatured polypeptides (201). PDI increases both the rate and efficiency of proper folding and export of proteins that contain disulfide bonds (195, 202). PDI1 is a gene essential for viability in yeast (203). There are different opinions regarding the significance of this observation. First, critical to the disulfide isomerase catalytic activity are two -CXXC- motifs. However, polypeptide binding by PDI has been reported not to involve these motifs (204), and PDI also has been shown to exhibit the potential for assisting folding of proteins that do not possess disulfide bonds (205). Further, certain deletions in PDI that leave residual disulfide isomerase activity are nevertheless lethal, while cells carrying a variant PDI in which both-cghcactive sites are disrupted (i.e., no measurable isomerase activity in vitro) remain viable (206) and able to assist in protein folding (207). On the other hand, the ER lumen appears to have other proteins (discussed further, below) that can function as disulfide isomerases in the absence of PDI1 enzyme activity (208). With this in mind, it is interesting that Escherichia coli thioredoxin can complement null mutants of yeast PDI only if thioredoxin is mutated to contain a reactive CXXC motif (209). These and similar studies have led some to conclude that the essential function of PDI is in fact to unscramble nonnative disulfide bonds (210). Nevertheless, PDI is recognized to be multifunctional; it is for instance a wellrecognized subunit of the prolyl-hydroxylase complex that is involved in collagen synthesis (211), and it heterodimerizes with the 97-kDa subunit of the microsomal triglyceride transfer protein complex (212). Peptidylprolyl isomerase (PPI) catalyzes cis-trans isomerization of proline side chains, and enhances the rate of protein folding in vitro (213). PPIs are also termed immunophilins and are comprised of cyclophilins (which bind the immunosuppressant cyclosporin A) and FKBPs (which bind the immunosuppressant FK506). The ER luminal FKBPs are transcriptionally regulated with other members of the unfolded protein response (149, 214). E. ERp72 and ER60 ERp72, one of the more recently described luminal chaperones (215), is also a calcium-binding protein in the ER (135, 208) and is a member of the PDI superfamily (see above). Although ERp72 is not an essential gene product, its overexpression can rescue nonviable cells deficient for PDI (216). Indeed, ERp72 contains three copies of the -CXXC- active site motif found in PDI (215). While it seems plausible that ERp72 may be able to exhibit limited PDI-like activity (208, 217), it also may be that its ability to rescue cells lacking PDI could be associated with the reported ability of ERp72 to assist in the degradation of proteins that cannot fold in the ER (218). ERp72 has been shown to interact with misfolded versions of exportable proteins (33, 219, 220), but evidence for its interaction with normal protein folding intermediates in vivo is not well established (143). Nevertheless, ERp72 is clearly regulated with other proteins exhibiting the ER unfolded protein response (196, 221). ER60/calregulin, which also contains thioredoxin-like sequences and thus shares homology with ERp72 and PDI, has been implicated in the ER-associated degradation of misfolded proteins (222). Although initially hypothesized to be a phosphoinositide-specific phospholipase C (223), this has not been independently confirmed. By contrast, although additional study is clearly needed, some evidence for a thioldependent protease activity of ER60 is accumulating (224). Moreover, it has recently been reported that processing of N-linked carbohydrates in exportable protein substrates
9 April, 1998 ENDOCRINE ERSDs 181 may be required for their association with ER60 (225), which has been proposed to act in conjunction with either calnexin or calreticulin (226). F. HSP47 Heat shock protein 47 (HSP47) is a collagen-specific stress protein that performs chaperone function during folding and assembly of newly synthesized procollagen molecules (227). Because type I procollagen forms a triple helix beginning at the carboxy terminus (see Section IV.C), much of its folding must occur after translocation of the nascent chain has been completed. Early association of HSP47 is facilitated by its binding to the amino-terminal globular domain of the collagen propeptide (228), but the chaperone appears to remain associated throughout most, if not all, subsequent stages of tertiary structural maturation (229, 230). Although it may act in concert with other, more ubiquitous ER chaperones (144, 231), the services of HSP47 appear to be unique to cells secreting collagen and its homologs (232). Because of this, HSP47 falls into a gray area of protein-specific ER chaperones, as is the case with the microsomal triglyceride transfer protein (see Fig. 1 and Section IV.D, below). G. Molecular escorts: pro-peptides, transport subunits, receptor-associated protein (RAP), and 7B2 Figure 1 provides a schematic to categorize a number of additional polypeptides that provide helper function to the protein export pathway, yet should nevertheless be distinguished from conventional ER chaperones. These include the propeptides of many polypeptide hormones, transport subunits, and a subgroup of proteins for which we propose to adopt the name molecular escorts (233). There are growing numbers of examples of proteins in each of these subgroups, but we mention only one or two representative examples. We emphasize that these distinctions may change over time, as molecular information about the roles of helper proteins in the folding pathways of individual proteins becomes clearer. Importantly, not all polypeptides that participate in the folding and trafficking of exportable proteins are ER residents. Specifically, the propeptide regions of many exportable proteins themselves serve primarily a structural, rather than a functional, role (234). Thus, folding in the absence of the propeptide, or in the presence of a mutated propeptide, may perturb the conformational maturation and ER export of polypeptide hormone precursors (235). Such proregions have been referred to as intramolecular chaperones, but a distinction should be made between this idea and classic ER chaperones (Fig. 1) which (except under unusual circumstances) are not transported down the secretory pathway, and whose turnover in the cell is far slower, and which routinely associate with folding intermediates of more than one kind of substrate protein. Not unlike the situation with the propeptides are subunits of oligomeric proteins that function primarily in polypeptide transport and stabilization, while playing a relatively minor role in subsequent biological activity. A case in point is the -subunit of the glycoprotein hormones (LH, FSH, CG, TSH), which combines noncovalently with -subunit in the ER (236). Both subunits must be partially folded before subunit assembly (64, ), and the specific regions used by and -subunits to combine have begun to be mapped ( ). When expressed by themselves, -subunits tend to be relatively poorly secreted, whereas when coexpressed with, a much higher fraction of is secreted, as heterodimers (244). Although a heterodimeric structure (and therefore, the presence of a subunit) is needed for biological activity (245), the evidence indicates that the -subunit, which is common to all of these hormones and is therefore unlikely to provide biological specificity, plays an important role in export of the different glycoprotein hormones, in particular for FSH and for TSH (246). Another subgroup of molecules that should be distinguished from conventional ER chaperones is represented by the molecular escorts (233). For illustrative purposes, we review only two members of this subgroup. RAP is a 40 kda polypeptide which interacts shortly after the biosynthesis of low-density lipoprotein receptor (LDLR)- related protein (LRP), and RAP travels with LRP molecules, having the potential to remain associated on the cell surface (247). By itself, RAP is a protein that is believed to be retained within the ER (248). Further, LRP in the absence of RAP is also defective for ER exit (233). However RAP association with LRP allows both partners to exit the ER (249), presumably by maintaining a given LRP in a favorable conformation (250) and by preventing premature association of LRP ligands in the ER (251), which could lead to receptor retention and/or degradation. Instead, dissociation of RAP, when it occurs, takes place after the proteins have reached the cell surface, where LRP ligands can displace the escort. 7B2 exhibits restrictive expression in neuroendocrine tissues, where it is synthesized as a 25-kDa precursor protein and is released from the regulated secretory pathway as processed proteolytic fragments. The carboxy-terminal domain of the pro7b2 protein is responsible for inhibiting the prohormone processing activity of prohormone convertase 2 (PC2) (252, 253), while the larger N-terminal portion of processed 7B2 contains independent stimulatory actions on the catalytic activity of PC2 (254). Evidently, several different domains of the pro-7b2 protein may interact with the proform of PC2 (255). Recently, it was determined that pro-7b2 and pro-pc2 assemble (in a 1:1 stoichiometry) in the ER of neuroendocrine secretory cells, and this assembly leaves the ER together (256) before dissociation in the most distal portions of the Golgi complex (257, 258). Because pro-pc2 may be transported through the secretory pathway more slowly or less efficiently in the absence of pro-7b2 (259), pro-7b2 has been called a chaperone (256) but may be better viewed as a molecular escort. Such a distinction also represents a gray area since recombinant 7B2 may be able to associate with at least one other unrelated protein, based on in vitro studies (260). III. Models of ER to Golgi Traffic Influence Models of Quality Control Newly synthesized plasma membrane proteins and secretory proteins must enter or cross the ER membrane in a
Protein Trafficking in the Secretory and Endocytic Pathways
Protein Trafficking in the Secretory and Endocytic Pathways The compartmentalization of eukaryotic cells has considerable functional advantages for the cell, but requires elaborate mechanisms to ensure
More informationProtein sorting (endoplasmic reticulum) Dr. Diala Abu-Hsasan School of Medicine
Protein sorting (endoplasmic reticulum) Dr. Diala Abu-Hsasan School of Medicine dr.abuhassand@gmail.com An overview of cellular components Endoplasmic reticulum (ER) It is a network of membrane-enclosed
More informationTRANSPORT PROCESSES. 1b. moving proteins into membranes and organelles
1b. moving proteins into membranes and organelles SLIDE 1 A typical mammalian cell contains up to 10,000 different kinds of proteins. The vast majority of these proteins are synthesized by cytosolic ribosomes,
More informationIntracellular Compartments and Protein Sorting
Intracellular Compartments and Protein Sorting Intracellular Compartments A eukaryotic cell is elaborately subdivided into functionally distinct, membrane-enclosed compartments. Each compartment, or organelle,
More informationMolecular Cell Biology Problem Drill 16: Intracellular Compartment and Protein Sorting
Molecular Cell Biology Problem Drill 16: Intracellular Compartment and Protein Sorting Question No. 1 of 10 Question 1. Which of the following statements about the nucleus is correct? Question #01 A. The
More informationGlycoprotein Maturation and Quality Control in the Endoplasmic Reticulum Dr. Daniel Hebert
Glycoprotein Maturation and Quality Control in the Endoplasmic Reticulum Department of Biochemistry and Molecular Biology University of Massachusetts, USA 1 Intracellular protein trafficking Plasma membrane
More information2013 John Wiley & Sons, Inc. All rights reserved. PROTEIN SORTING. Lecture 10 BIOL 266/ Biology Department Concordia University. Dr. S.
PROTEIN SORTING Lecture 10 BIOL 266/4 2014-15 Dr. S. Azam Biology Department Concordia University Introduction Membranes divide the cytoplasm of eukaryotic cells into distinct compartments. The endomembrane
More informationEndoplasmic Reticulum
Endoplasmic Reticulum What s ER? How is ER? Why is ER? definition description functions Nissl s bodies neurons Berg s bodies hepatocytes Organelle structure histocytochemical evidences Ergastoplasm pancreatic
More informationCell Quality Control. Peter Takizawa Department of Cell Biology
Cell Quality Control Peter Takizawa Department of Cell Biology Cellular quality control reduces production of defective proteins. Cells have many quality control systems to ensure that cell does not build
More informationPosttranslational Modification and Targeting of Proteins
Posttranslational Modification and Targeting of Proteins Graduate Biochemistry Term 2/2016 Assist. Prof. Dr. Panida Khunkaewla School of Chemistry, Institute of Science Suranaree University of Technology
More informationSummary of Endomembrane-system
Summary of Endomembrane-system 1. Endomembrane System: The structural and functional relationship organelles including ER,Golgi complex, lysosome, endosomes, secretory vesicles. 2. Membrane-bound structures
More informationMoving Proteins into Membranes and Organelles
13 Moving Proteins into Membranes and Organelles Review the Concepts 1. In eukaryotes, protein translocation across the endoplasmic reticulum (ER) membrane is most commonly cotranslational; it can also
More informationPractice Exam 2 MCBII
1. Which feature is true for signal sequences and for stop transfer transmembrane domains (4 pts)? A. They are both 20 hydrophobic amino acids long. B. They are both found at the N-terminus of the protein.
More informationHomework Hanson section MCB Course, Fall 2014
Homework Hanson section MCB Course, Fall 2014 (1) Antitrypsin, which inhibits certain proteases, is normally secreted into the bloodstream by liver cells. Antitrypsin is absent from the bloodstream of
More informationChapter 6. Antigen Presentation to T lymphocytes
Chapter 6 Antigen Presentation to T lymphocytes Generation of T-cell Receptor Ligands T cells only recognize Ags displayed on cell surfaces These Ags may be derived from pathogens that replicate within
More information1. endoplasmic reticulum This is the location where N-linked oligosaccharide is initially synthesized and attached to glycoproteins.
Biology 4410 Name Spring 2006 Exam 2 A. Multiple Choice, 2 pt each Pick the best choice from the list of choices, and write it in the space provided. Some choices may be used more than once, and other
More information1. This is the location where N-linked oligosaccharide is initially synthesized and attached to glycoproteins.
Biology 4410 Name Spring 2006 Exam 2 A. Multiple Choice, 2 pt each Pick the best choice from the list of choices, and write it in the space provided. Some choices may be used more than once, and other
More informationendomembrane system internal membranes origins transport of proteins chapter 15 endomembrane system
endo system chapter 15 internal s endo system functions as a coordinated unit divide cytoplasm into distinct compartments controls exocytosis and endocytosis movement of molecules which cannot pass through
More informationREGULATION OF ENZYME ACTIVITY. Medical Biochemistry, Lecture 25
REGULATION OF ENZYME ACTIVITY Medical Biochemistry, Lecture 25 Lecture 25, Outline General properties of enzyme regulation Regulation of enzyme concentrations Allosteric enzymes and feedback inhibition
More informationIntracellular vesicular traffic. B. Balen
Intracellular vesicular traffic B. Balen Three types of transport in eukaryotic cells Figure 12-6 Molecular Biology of the Cell ( Garland Science 2008) Endoplasmic reticulum in all eucaryotic cells Endoplasmic
More information1. to understand how proteins find their destination in prokaryotic and eukaryotic cells 2. to know how proteins are bio-recycled
Protein Targeting Objectives 1. to understand how proteins find their destination in prokaryotic and eukaryotic cells 2. to know how proteins are bio-recycled As a protein is being synthesized, decisions
More informationZool 3200: Cell Biology Exam 4 Part I 2/3/15
Name: Key Trask Zool 3200: Cell Biology Exam 4 Part I 2/3/15 Answer each of the following questions in the space provided, explaining your answers when asked to do so; circle the correct answer or answers
More informationPROTEIN TRAFFICKING. Dr. SARRAY Sameh, Ph.D
PROTEIN TRAFFICKING Dr. SARRAY Sameh, Ph.D Overview Proteins are synthesized either on free ribosomes or on ribosomes bound to endoplasmic reticulum (RER). The synthesis of nuclear, mitochondrial and peroxisomal
More informationBIOL 4374/BCHS 4313 Cell Biology Exam #2 March 22, 2001
BIOL 4374/BCHS 4313 Cell Biology Exam #2 March 22, 2001 SS# Name This exam is worth a total of 100 points. The number of points each question is worth is shown in parentheses. Good luck! 1. (2) In the
More informationChapter 13 Moving Proteins into Membranes and Organelles
Chapter 13 Moving Proteins into Membranes and Organelles Chapter 13 Moving Proteins into Membranes and Organelles 13.1 Targeting Proteins To and Across the ER Membrane 13.2 Insertion of Membrane Proteins
More informationMCB II MCDB 3451 Exam 1 Spring, minutes, close everything and be concise!
MCB II MCDB 3451 Exam 1 Spring, 2016 50 minutes, close everything and be concise! Name ID NOTE: QUESTIONS ARE NOT ALL WORTH THE SAME POINTS Total (100) Grade EXAM 1, 2016 MCBII Name 1. Which is UNIQUE
More informationIntroduction and protein sorting
Introduction and protein sorting Membrane proteins Major components of cells Nucleic acids Carbohydrates Proteins Lipids (50% of mass of plasma membranes, 30% of mitochondrial membranes, 80% of myelin
More informationSignal Transduction Cascades
Signal Transduction Cascades Contents of this page: Kinases & phosphatases Protein Kinase A (camp-dependent protein kinase) G-protein signal cascade Structure of G-proteins Small GTP-binding proteins,
More informationChapter 31. Completing the Protein Life Cycle: Folding, Processing and Degradation. Biochemistry by Reginald Garrett and Charles Grisham
Chapter 31 Completing the Protein Life Cycle: Folding, Processing and Degradation Biochemistry by Reginald Garrett and Charles Grisham Essential Question How are newly synthesized polypeptide chains transformed
More informationAmino Acids and Proteins (2) Professor Dr. Raid M. H. Al-Salih
Amino Acids and Proteins (2) Professor Dr. Raid M. H. Al-Salih 1 Some important biologically active peptides 2 Proteins The word protein is derived from Greek word, proteios which means primary. As the
More informationMCB130 Midterm. GSI s Name:
1. Peroxisomes are small, membrane-enclosed organelles that function in the degradation of fatty acids and in the degradation of H 2 O 2. Peroxisomes are not part of the secretory pathway and peroxisomal
More informationThe endoplasmic reticulum is a network of folded membranes that form channels through the cytoplasm and sacs called cisternae.
Endoplasmic reticulum (ER) The endoplasmic reticulum is a network of folded membranes that form channels through the cytoplasm and sacs called cisternae. Cisternae serve as channels for the transport of
More informationMolecular Trafficking
SCBM 251 Molecular Trafficking Assoc. Prof. Rutaiwan Tohtong Department of Biochemistry Faculty of Science rutaiwan.toh@mahidol.ac.th Lecture outline 1. What is molecular trafficking? Why is it important?
More informationMOLECULAR CELL BIOLOGY
1 Lodish Berk Kaiser Krieger scott Bretscher Ploegh Matsudaira MOLECULAR CELL BIOLOGY SEVENTH EDITION CHAPTER 13 Moving Proteins into Membranes and Organelles Copyright 2013 by W. H. Freeman and Company
More informationLecture 15. Signal Transduction Pathways - Introduction
Lecture 15 Signal Transduction Pathways - Introduction So far.. Regulation of mrna synthesis Regulation of rrna synthesis Regulation of trna & 5S rrna synthesis Regulation of gene expression by signals
More informationMolecular Cell Biology 5068 In Class Exam 1 October 3, 2013
Molecular Cell Biology 5068 In Class Exam 1 October 3, 2013 Exam Number: Please print your name: Instructions: Please write only on these pages, in the spaces allotted and not on the back. Write your number
More informationProtein Modification Overview DEFINITION The modification of selected residues in a protein and not as a component of synthesis
Lecture Four: Protein Modification & Cleavage [Based on Chapters 2, 9, 10 & 11 Berg, Tymoczko & Stryer] (Figures in red are for the 7th Edition) (Figures in Blue are for the 8th Edition) Protein Modification
More informationName: Multiple choice questions. Pick the BEST answer (2 pts ea)
Exam 1 202 Oct. 5, 1999 Multiple choice questions. Pick the BEST answer (2 pts ea) 1. The lipids of a red blood cell membrane are all a. phospholipids b. amphipathic c. glycolipids d. unsaturated 2. The
More informationChapter 10. Regulatory Strategy
Chapter 10 Regulatory Strategy Regulation of enzymatic activity: 1. Allosteric Control. Allosteric proteins have a regulatory site(s) and multiple functional sites Activity of proteins is regulated by
More informationLecture Readings. Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell
October 26, 2006 1 Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs
More informationMolecular Cell Biology - Problem Drill 17: Intracellular Vesicular Traffic
Molecular Cell Biology - Problem Drill 17: Intracellular Vesicular Traffic Question No. 1 of 10 1. Which of the following statements about clathrin-coated vesicles is correct? Question #1 (A) There are
More informationEndomembrane system 11/1/2018. Endomembrane System. Direct physical continuity. Transfer of membrane segments as vesicles. Outer Nuclear envelope
Endomembrane system Endomembrane System Outer Nuclear envelope Direct physical continuity Transfer of membrane segments as vesicles Endoplasmic reticulum BUT membranes are not identical in structure and
More informationG-Protein Signaling. Introduction to intracellular signaling. Dr. SARRAY Sameh, Ph.D
G-Protein Signaling Introduction to intracellular signaling Dr. SARRAY Sameh, Ph.D Cell signaling Cells communicate via extracellular signaling molecules (Hormones, growth factors and neurotransmitters
More informationPeptide hydrolysis uncatalyzed half-life = ~450 years HIV protease-catalyzed half-life = ~3 seconds
Uncatalyzed half-life Peptide hydrolysis uncatalyzed half-life = ~450 years IV protease-catalyzed half-life = ~3 seconds Life Sciences 1a Lecture Slides Set 9 Fall 2006-2007 Prof. David R. Liu In the absence
More informationCellular functions of protein degradation
Protein Degradation Cellular functions of protein degradation 1. Elimination of misfolded and damaged proteins: Environmental toxins, translation errors and genetic mutations can damage proteins. Misfolded
More informationPeter Walter, UCSF IRE1 Signaling Affects Cell Fate during the Unfolded Protein Response
Peter Walter, UCSF IRE1 Signaling Affects Cell Fate during the Unfolded Protein Response Jenn Hou Burke Group Literature Seminar November 19 th 2016 Protein Synthesis Pathway 4. Final Destination: proteins
More informationSheet #5 Dr. Mamoun Ahram 8/7/2014
P a g e 1 Protein Structure Quick revision - Levels of protein structure: primary, secondary, tertiary & quaternary. - Primary structure is the sequence of amino acids residues. It determines the other
More informationW I S S E N T E C H N I K L E I D E N S C H A F T MOL.911. Cell Engineering. u
1 W I S S E N T E C H N I K L E I D E N S C H A F T MOL.911 Cell Engineering u www.tugraz.at MOL.911 Molecular Biotechnology I 2 Cell Engineering General strategies: Knock out of specific genes - Gene
More informationC) You find that the Raf kinase is not constitutively active. What was necessary in the previous assay to show any Raf kinase activity?
PROBLEM SET 3 1. You have obtained immortalized liver cells from a patient who died of Wilson s disease, an inherited disorder of copper metabolism marked by neuronal degeneration and hepatic cirrhosis.
More informationGeneral information. Cell mediated immunity. 455 LSA, Tuesday 11 to noon. Anytime after class.
General information Cell mediated immunity 455 LSA, Tuesday 11 to noon Anytime after class T-cell precursors Thymus Naive T-cells (CD8 or CD4) email: lcoscoy@berkeley.edu edu Use MCB150 as subject line
More informationComplexity DNA. Genome RNA. Transcriptome. Protein. Proteome. Metabolites. Metabolome
DNA Genome Complexity RNA Transcriptome Systems Biology Linking all the components of a cell in a quantitative and temporal manner Protein Proteome Metabolites Metabolome Where are the functional elements?
More informationSignificance and Functions of Carbohydrates. Bacterial Cell Walls
Biochemistry 462a - Carbohydrate Function Reading - Chapter 9 Practice problems - Chapter 9: 2, 4a, 4b, 6, 9, 10, 13, 14, 15, 16a, 17; Carbohydrate extra problems Significance and Functions of Carbohydrates
More informationCELL BIOLOGY - CLUTCH CH INTRACELLULAR PROTEIN TRANSPORT.
!! www.clutchprep.com CONCEPT: MEMBRANE ENCLOSED ORGANELLES Table of eukaryotic organelles and their functions Organelle Function % volume of cell Cytosol Aqueous fluid where metabolic pathways and chemical
More informationAP Biology
Tour of the Cell (1) 2007-2008 Types of cells Prokaryote bacteria cells - no organelles - organelles Eukaryote animal cells Eukaryote plant cells Cell Size Why organelles? Specialized structures - specialized
More informationChapter 9. Cellular Signaling
Chapter 9 Cellular Signaling Cellular Messaging Page 215 Cells can signal to each other and interpret the signals they receive from other cells and the environment Signals are most often chemicals The
More informationReview of Biochemistry
Review of Biochemistry Chemical bond Functional Groups Amino Acid Protein Structure and Function Proteins are polymers of amino acids. Each amino acids in a protein contains a amino group, - NH 2,
More informationChapter 11: Enzyme Catalysis
Chapter 11: Enzyme Catalysis Matching A) high B) deprotonated C) protonated D) least resistance E) motion F) rate-determining G) leaving group H) short peptides I) amino acid J) low K) coenzymes L) concerted
More informationN-Linked Carbohydrates Act as Lumenal Maturation and Quality Control Protein Tags
Copyright 2004 by Humana Press Inc. All rights of any nature whatsoever reserved. 1085-9195/04/41:113 137/$25.00 REVIEW ARTICLE N-Linked Carbohydrates Act as Lumenal Maturation and Quality Control Protein
More informationOctober 26, Lecture Readings. Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell
October 26, 2006 Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs
More informationIn and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases
Physiol Rev 87: 1377 1408, 2007; doi:10.1152/physrev.00050.2006. In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases DANIEL N. HEBERT AND MAURIZIO MOLINARI Institute
More informationQUALITY CONTROL IN THE ENDOPLASMIC RETICULUM
QUALITY CONTROL IN THE ENDOPLASMIC RETICULUM Lars Ellgaard and Ari Helenius The endoplasmic reticulum (ER) has a quality-control system for proof-reading newly synthesized proteins, so that only native
More informationMolecular Cell Biology 5068 In class Exam 1 October 2, Please print your name: Instructions:
Molecular Cell Biology 5068 In class Exam 1 October 2, 2012 Exam Number: Please print your name: Instructions: Please write only on these pages, in the spaces allotted and not on the back. Write your number
More informationAmino acids. Side chain. -Carbon atom. Carboxyl group. Amino group
PROTEINS Amino acids Side chain -Carbon atom Amino group Carboxyl group Amino acids Primary structure Amino acid monomers Peptide bond Peptide bond Amino group Carboxyl group Peptide bond N-terminal (
More informationProteins. Bởi: OpenStaxCollege
Proteins Bởi: OpenStaxCollege Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory,
More information17/01/2017. Protein trafficking between cell compartments. Lecture 3: The cytosol. The mitochondrion - the power plant of the cell
ell biology 2017 version 13/1 2017 ote endosome vs lysosome handout Lecture 3: Text book Alberts et al.: hapter 12-14 (Topics covered by the lecture) A lot of reading! Focus on principles ell Biology interactive
More informationThe cell biology of major histocompatibility complex class I assembly: towards a molecular understanding
Tissue Antigens ISSN 0001-2815 REVIEW ARTICLE The cell biology of major histocompatibility complex class I assembly: towards a molecular understanding A. van Hateren 1,3, E. James 1,3, A. Bailey 1,2,3,
More informationProblem Set 5, 7.06, Spring of 13
Problem Set 5, 7.06, Spring 2003 1 of 13 1. In order to please your demanding thesis advisor, you've completed an extensive fractionation and biochemical purification of proteins localized to the mitochondria,
More informationSection 6. Junaid Malek, M.D.
Section 6 Junaid Malek, M.D. The Golgi and gp160 gp160 transported from ER to the Golgi in coated vesicles These coated vesicles fuse to the cis portion of the Golgi and deposit their cargo in the cisternae
More informationCELLS. Cells. Basic unit of life (except virus)
Basic unit of life (except virus) CELLS Prokaryotic, w/o nucleus, bacteria Eukaryotic, w/ nucleus Various cell types specialized for particular function. Differentiation. Over 200 human cell types 56%
More informationLecture Series 2 Macromolecules: Their Structure and Function
Lecture Series 2 Macromolecules: Their Structure and Function Reading Assignments Read Chapter 4 (Protein structure & Function) Biological Substances found in Living Tissues The big four in terms of macromolecules
More informationSignificance of the MHC
CHAPTER 8 Major Histocompatibility Complex (MHC) What is MHC? HLA H-2 Minor histocompatibility antigens Peter Gorer & George Sneell (1940) - MHC molecules were initially discovered during studies aimed
More informationStructure of proteins
Structure of proteins Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry I Philadelphia University Faculty of pharmacy Structure of proteins The 20 a.a commonly found
More informationCopyright 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
Concept 5.4: Proteins have many structures, resulting in a wide range of functions Proteins account for more than 50% of the dry mass of most cells Protein functions include structural support, storage,
More informationProtein and membrane trafficking
Protein and membrane trafficking Lodish et al, 2000 The secretory pathway cisternal progression Lodish et al, 2000 Cells can be specialized for the secretion of specific proteins Lodish et al, 2000 The
More informationLecture Series 2 Macromolecules: Their Structure and Function
Lecture Series 2 Macromolecules: Their Structure and Function Reading Assignments Read Chapter 4 (Protein structure & Function) Biological Substances found in Living Tissues The big four in terms of macromolecules
More informationVesicle Transport. Vesicle pathway: many compartments, interconnected by trafficking routes 3/17/14
Vesicle Transport Vesicle Formation Curvature (Self Assembly of Coat complex) Sorting (Sorting Complex formation) Regulation (Sar1/Arf1 GTPases) Fission () Membrane Fusion SNARE combinations Tethers Regulation
More informationQ1: Circle the best correct answer: (15 marks)
Q1: Circle the best correct answer: (15 marks) 1. Which one of the following incorrectly pairs an amino acid with a valid chemical characteristic a. Glycine, is chiral b. Tyrosine and tryptophan; at neutral
More informationProtein modification. Protein Modification, targeting and degradation. Structure of Collagen (continued) Structure of Collagen
Protein modification Protein Modification, targeting and degradation Proteins undergo a variety of modifications that are critical for function. There are numerous amino acid modifications such as collagen.
More informationA. Lipids: Water-Insoluble Molecules
Biological Substances found in Living Tissues Lecture Series 3 Macromolecules: Their Structure and Function A. Lipids: Water-Insoluble Lipids can form large biological molecules, but these aggregations
More information1) DNA unzips - hydrogen bonds between base pairs are broken by special enzymes.
Biology 12 Cell Cycle To divide, a cell must complete several important tasks: it must grow, during which it performs protein synthesis (G1 phase) replicate its genetic material /DNA (S phase), and physically
More informationAn aldose contains an aldehyde functionality A ketose contains a ketone functionality
RCT Chapter 7 Aldoses and Ketoses; Representative monosaccharides. (a)two trioses, an aldose and a ketose. The carbonyl group in each is shaded. An aldose contains an aldehyde functionality A ketose contains
More informationZool 3200: Cell Biology Exam 4 Part I 2/3/15
Name: Trask Zool 3200: Cell Biology Exam 4 Part I 2/3/15 Answer each of the following questions in the space provided, explaining your answers when asked to do so; circle the correct answer or answers
More informationCell Biology from an Immune Perspective
Harvard-MIT Division of Health Sciences and Technology HST.176: Cellular and Molecular Immunology Course Director: Dr. Shiv Pillai Cell Biology from an Immune Perspective In this lecture we will very briefly
More informationAntigen presenting cells
Antigen recognition by T and B cells - T and B cells exhibit fundamental differences in antigen recognition - B cells recognize antigen free in solution (native antigen). - T cells recognize antigen after
More informationThe Structure and Function of Large Biological Molecules Part 4: Proteins Chapter 5
Key Concepts: The Structure and Function of Large Biological Molecules Part 4: Proteins Chapter 5 Proteins include a diversity of structures, resulting in a wide range of functions Proteins Enzymatic s
More informationTuesday, Sept. 14, Is an enzyme a rigid system?
Tuesday, Sept. 14, Is an enzyme a rigid system? Early researchers thought of enzymes as rigid entities, recognizing their substrates the way a lock would recognize a key. Today's researchers, however,
More informationINTERACTION DRUG BODY
INTERACTION DRUG BODY What the drug does to the body What the body does to the drug Receptors - intracellular receptors - membrane receptors - Channel receptors - G protein-coupled receptors - Tyrosine-kinase
More informationMechanism of Vesicular Transport
Mechanism of Vesicular Transport Transport vesicles play a central role in the traffic of molecules between different membrane-enclosed enclosed compartments. The selectivity of such transport is therefore
More informationObjective: You will be able to explain how the subcomponents of
Objective: You will be able to explain how the subcomponents of nucleic acids determine the properties of that polymer. Do Now: Read the first two paragraphs from enduring understanding 4.A Essential knowledge:
More informationPAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 22, Mechanism of enzyme catalyst reaction (I) Chymotrypsin
Subject Paper No and Title 16 Bio-organic and Biophysical Module No and Title 22 Mechanism of Enzyme Catalyzed reactions I Module Tag CHE_P16_M22 Chymotrypsin TABLE OF CONTENTS 1. Learning outcomes 2.
More informationChapter 13: Vesicular Traffic
Chapter 13: Vesicular Traffic Know the terminology: ER, Golgi, vesicle, clathrin, COP-I, COP-II, BiP, glycosylation, KDEL, microtubule, SNAREs, dynamin, mannose-6-phosphate, M6P receptor, endocytosis,
More informationReceptor mediated Signal Transduction
Receptor mediated Signal Transduction G-protein-linked receptors adenylyl cyclase camp PKA Organization of receptor protein-tyrosine kinases From G.M. Cooper, The Cell. A molecular approach, 2004, third
More informationCell Communication. Local and Long Distance Signaling
Cell Communication Cell to cell communication is essential for multicellular organisms Some universal mechanisms of cellular regulation providing more evidence for the evolutionary relatedness of all life
More informationExams written in pencil or erasable ink will not be re-graded under any circumstances.
Biochemistry 461, Section I May 21, 1998 Final Exam Prof. Jason D. Kahn Your Printed ame: Your SS#: Your Signature: You have 120 minutes for this exam. The exam has 7 questions, worth 200 points. Do all
More information/ The following functional group is a. Aldehyde c. Carboxyl b. Ketone d. Amino
Section A: Multiple Choice Select the answer that best answers the following questions. Please write your selected choice on the line provided, in addition to circling the answer. /25 1. The following
More information6.5 Enzymes. Enzyme Active Site and Substrate Specificity
180 Chapter 6 Metabolism 6.5 Enzymes By the end of this section, you will be able to: Describe the role of enzymes in metabolic pathways Explain how enzymes function as molecular catalysts Discuss enzyme
More informationChapter 1: Vesicular traffic. Biochimica cellulare parte B 2017/18
Chapter 1: Vesicular traffic Biochimica cellulare parte B 2017/18 Major Protein-sorting pathways in eukaryotic cells Secretory and endocytic pathways Unifying principle governs all protein trafficking
More informationChem Exam 2 (A) Name
Chem 4511 Exam 2 (A) Name No credit will be given for answers (or work) that are on the backsides of the pages. You may use the backsides as scratch paper, but put all of your answers on the front sides.
More informationEndocrine pharmacology (3)
بسم رلا هللا Endocrine pharmacology (3) Natural hormone characterized by short of action : a lot of them ineffective orally ( for example ), but when we give it from outside it enters the body exactly
More informationCell Biology Lecture 9 Notes Basic Principles of cell signaling and GPCR system
Cell Biology Lecture 9 Notes Basic Principles of cell signaling and GPCR system Basic Elements of cell signaling: Signal or signaling molecule (ligand, first messenger) o Small molecules (epinephrine,
More information