Autophagic-lysosomal and mitochondrial sequestration of [I 4C]sucrose

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1 Eui. J Biochem 153, (1985) ( FEBS 1985 Autophagic-lysosomal and mitochondrial sequestration of [I 4C]sucrose Density gradient distribution of sequestered radioactivity Hclge TOLI.ESHAUG and Per 0. SEGLEN Department of Tissue Culture, Norsk Hydro's Institute for C,ancer Research, The Norwegian Radium Hospital, Oslo (Received August 7, 1985) - EJB ['4C]Sucrose, inlroduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 C. The sedimentation characteristics of particle-associated [''C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. tsopycnic banding in isotonic iiietrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak ( I0 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.I5 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of ['4C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic Sequestration of ['4C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy. Autophagic sequcstration, the first step in the autophagiclysosomal pathway for degradation of proteins and other intracellular macromolecules, can be approached experimentally by measuring the transfer of an inert probe (e.g. [14C]sucrose) from cytosol to sedimentable cell structures. The problem of getting the probe into the cell across a normally impermeable plasma membrane can be overcome by using various techniques such as pipette microinjection [I], crythrocyte ghost fusion [2] or reversible electropermeabilization [3, 41. By means of the latter method we have been able to show that isolated rat hepatocytes sequester cytosolic ['4C]sucrose at a considerable rate, and that the sequestration is sensitive to autophagy inhibitors like 3-methyladenine and amino acids [4]. However, our experiments indicated a heterogeneity in sequestration mechanisms, which prompted us to examine the subcellular localization of the sequestering activities. In this communication we present evidence for a mitochondrial [14C]sucrose uptake mechanism in addition to autophagiclysosomal sequestration. A preliminary report of some aspects of this work was given in [5]. MATERIALS AND METHODS Reagents [14C]Sucro~e(uniformly labelled, 554 Ci/mol; 210 mci/ ml) was obtained from Amersham International (Bucks, UK). Correspondence to P. 0. Seglen, Avdeling for Vevsdyrkning, Norsk Hydro's Institutt for Kreftforskning, Det Norske Radiumhospital, Montebello, Oslo 3, Norway Enzymes. Acid phosphalase (EC ); cytochrome oxidase (EC 1.Y.3.1); hexosaminidase or N-acetyl-[Gglucosaminidase (EC ). 251-Asialoorosomucoid (100 Ci/mmol; 4.5 pci/ml) was synthesized as described elsewhere [6, 71. Metrizamide was purchased from Nyegaard & Co. (Oslo, Norway) and 3-methyladenine (6-amino-3-methylpurine) from Fluka AG (Buchs, Switzerland). Other biochemicals were from Sigma Chem. Co. (St Louis, MO, USA). Isolution and incubation oj heppcitocytes Isolated hepatocytes were prepared from the liver of 18- h-starved male Wistar rats, g, by collagenase perfusion [8]. The cells were kept in suspension buffer [8] fortified with pyruvate (20 mm) and Mg2+ (2mM). Incubations were done in 10-cm petri dishes (5-10 ml cell suspension in each dish), either stationary at 0' C or gently shaking on a tilting platform at 37'C. Relatively high cell concentrations (z 150 mg wet weight/ml; i.e. 20 million cells/ ml) are used with this type of incubation; the autophagiclysosomal activity is therefore somewhat submaximal owing to rapid amino acid conditioning of the medium 191. Electropermeabilization, [ ''C/sucrose louding and sequestration Hepatocytes in suspension were electropernieabilized with five high-voltage pulses (2 kvjcm) as previously described [4]. The permeabilized cells were diffusion-loaded with ['4C]sucrose (5-10 pci/ml) for 1 h at O"C, then incubated 30 min at 37 C (still in the presence of [14C]sucrose) to achieve resealing. The cells (now non-permeable) were then washed 3 x with ice-cold wash buffer [8] to remove extracellular radioactivity, whereupon incubation at 37 C was resumed. During the latter incubation, generally for 2 h, sequestration of cytosolic ['4C]sucrose into sedimentable organelles took place.

2 224 Table 1. Subcellular fractionation of cell corpses by dqjerential centriftigation Hepatocytes were electroloaded with ['4C]sucrose and incubated for 2.5 h at 37 "C. After electrodisruption, the purified cell corpscs wcre homogenized, and subcellular fractions were prepared by differential centrifugation. Each fraction (with the exception of the last) was washed and recentrifuged once. The amount of ['4C]sucrose associated with cell corpses increased 2.7-fold during the last 2 h of incubation. Each value is the mean f SE of three experiments Fraction x Sedi- Protein Acid ['4C]Sucrose Increase in Ratio designation mentation phosphatase sequestration of J/.X force (4 ['4C]sucrose h ~ ~ g. min Cell corpses - Large particles 8 Medium-sized particles 35 Small particles 300 Very small particles Soluble fraction - Recovery 'YO total at 2.5 h f f f f f f f f f 5.4 tv) % total ~ _ -fold f f I f f f % _ & f Electrodisruption, cell corpse purijkation and homogenization After incubation, the cells were washed and resuspended in ice-cold 10% sucrose (electrolyte-free), and electrodisrupted in 1 -ml portions at 37 C [3,4]. The cell corpses (structurally intact cell bodies with a disrupted plasma membrane) were purified by centrifugation through metrizamide/sucrose cushions 13i, resuspended in 0.25 sucrose/2 mm Hepes (PH 7.4), pooled and homogenized with a hand-operated homogenizer to a final homogenate concentration of about 10% (wet weight/volume) [lo]. Dijferentiul centrifugation Homogenates were fractionated by centrifugation (OOC) at stepwise increasing speeds, by a scheme roughly similar to that of de Duve et al. [lo, 111, into a large ('nuclear'), mediumsized ('mitochondrial'), small ('light-mitochondriai/lysosomal') and very small-sized ('microsomal') particle fraction as well as a soluble ('postmicrosomal') fraction. All sediments (except the last) were resuspended and recentrifuged once and the supernatants from each step were pooled before further processing. The sedimentation forces are given in Table 1. Cross-contamination between fractions was rather variable, in particular with respect to the acid phosphatase content of the medium-sized particle fraction sedimenting at x g. min. While on an average about one-third of the enzyme was found in this fraction, the experiment displayed in Fig. 5 succeeded in preparing an almost acid-phosphatase-free mitochondria1 fraction. It should be noted that, since the homogenates were prepared from cytosol-free cell corpses, the postmicrosomal (final) supernatant contains very little protein. Me tr izum ide gradient cen tr fuga tion Metrizamide/sucrose density gradients (0-40% metrizamide, w/v; M sucrose; both buffered with 2 mm Hepes, ph 7.3) were generated by diffusion between five layers of different density in horizontal tubes [lo]. 0.5 ml homogenate was layered on top of each of the near-linear 11 -ml gradients and overlayered with 1 ml 0.2 M buffered sucrose. The gradients were centrifuged (at OOC) for 30 min at rpm (3.4 x lo6 x g. min); both ['4C]sucrose and acid phosphatase were found to reach stable distributions within this time. The gradients were fractionated from the bottom into about twenty 12-drop (0.63-ml) fractions; the two or three uppermost fractions (containing the bulk of the nonsedimentable material) as well as the two bottom fractions (containing sedimented nuclei and variable amounts ofdebris) were pooled alld averaged in the graphic displays, The density of each fraction was calculated on the basis of refractive index (n), using an empirical formula developed for metrizamidei sucrose gradients: density (g/ml) = n x ) Enzyme assays Acid phosphatase was measured with 2-glycerophosphatc as substrate [12] in the presence of 0.5% Triton X-100 as previously described [13]. Hexosaminidase (N-acetyl-/Iglucosaminidase) was measured according to Barrett [12]. The cytochrome oxidase measurement was based on the method of Cooperstein and Lazarow [14], modified by using NaBH4 (2 mg/ml) to reduce the substrate. The solution was kept in a refrigerator for h, by which time all of the borohydride ions had decomposed. Sephadex gel filtration Samples (0.5 ml) of various markers (['4C]sucrose and [3H]glucose, 0.2 pci; blue dextran, 0.5 mg) or frozen/thawed peak fractions from metrizamide density gradients were applied to a 60-ml Sephadex (3-15 column, and eluted (with 0.9% NaCl containing 10 mm sucrose and 10 mm Hepcs, ph 7.3) at a speed of 25 ml/h. Column fractions (1 ml) were collected and analysed for radioactivity. Digitonin extraction For extraction of whole cells, digitonin was added to cell suspensions to a final concentration of 1 mg/ml. After 10 min at 0 "C the extracted cells were separated from the medium by centrifugation through metrizamide/sucrose cushions [3] and homogenized. For extraction of cell corpses, various concentrations of digitonin were used (standard concentration, 0.3 mgiml).

3 A A A - I I I, I I I I 225 Table 2. Lysosomal stability (acid phosphatase latency) in suhccliulur,fractions Hepatocytes were incubated and fractionated as in Table 1, and the activity of acid phosphatase measured by a 30-min assay in the presence and absence of0.5% Triton X-100 [13]. The latency is the activity without Triton expressed as a percentage of the activity with Triton, each value being the mean range of two experiments (or a single experiment in the case of the homogenate) Fraction designation Acid phosphatasc latency 0.5-h 2.5-h incubation incubation CENTRIFUGATION FORCE (g min lo3) Fig. 1. S~~dimentahiiit~i; uf (14CJ,s~~r~~e-.~~q~e~tering orgunellex [ I4C]- Sucrose-loaded hepatocytes were allowed to reseal for 0.5 h, washed and incubated for another 2 h at 37 C. Cytosol-free cell corpses were prepared at 0.5 h and 2.5 h, homogenized, and 1-ml aliquots of homogenate (2.5%, w/v) were centrifuged at 6200 xg, (4900 rpm) for various lengths of time. The supernatant was removed, and radioactiv- Soluble fraction ity and acid phosphatasc were measured in the sedimented pellet. (0) [ 4C]Sucrose at 0.5 h; (0)[ 4C]sucrose at 2.5 h; (A)[ 4C]sucrose h (net sequestration); (A) acid phosphatase. Values are expressed as percentages of the total in the homogenate; each value is the mean of two parallel homogenate aliquots After 10 min at O C, the cell corpses were resedimented and the supernatant taken for analysis. RESULTS Suhcellulur distribution ojsequestered 4C]sucrose We have previously shown that cell corpses, i.e. electrodisrupted cells centrifuged through metrizamide/sucrose cushions (to remove soluble cytosolic components), retain intact lysosomes as indicated by an essentially complete recovery of acid phosphatase [4]. Cell corpses, prepared from hepatocytes which have been electroinjected with [ 4C]sucrose and incubated for 2.5 hat 37 C, contain a considerable quantity of sequestered sucrose (z 15% of the total cell-associated radioactivity) as well as a background radioactivity (z 5% of total cell-associated radioactivity) of trapped cytosolic/ extracellular sucrose plus sucrose pinocytosed during the first 30-min resealing period, when the sugar is also present extracellularly (cf. Materials and Methods). Sedimentability. Homogenates prepared from cell corpses by the use of a hand-operated Dounce homogenizer contained 75-85% intact lysosomes (sedimentable acid phosphatase), indicating a limited extent of homogenization-induced damage (Fig. 1). The [14C]sucrose background at 30 min was only 40-50% sedimentable in the homogenates at the (relatively moderate) centrifugation forces used; an additional 10% (small pinosomes?) could be sedimented with a 100 times higher centrifugation force (results not shown). The remaining non-sedimentable radioactivity can proabably be accounted for by organelle disruption plus some carry-over of cytosolic/ extracellular [ 4C]sucrose [4]. The sedimentability of [14C]sucrose was much higher at 2.5 h, and by subtraction of the 30-min values it became evident that the sucrose sequestered during the incubation was almost 100% sedimentable (Fig. 1). The sucrose-sequestering organelles Electrodisrupted cells Cell corpses Cell corpse homogenate Large particles Medium-sized particles Small particles Very small particles 14.3 f * f f f * _ f f f i f t 0.3 would therefore seem to be, on average, less fragile than the average lysosome. D (Heren t ial centr zfugat ion. Sed imen ta ble particles can be roughly separated into different size classes by differential centrifugation, i.e. sedimentation at progressively increasing centrifugal forces. Table 1 shows the distributions of [ CIsucrose, acid phosphatase and protein in four particle fractions plus the final supernatant, prepared from the homogenized corpses of cells which had been sequestering [ 4C]sucrose for 2.5 h. The distributions of protein and acid phosphatase did not change significantly between 0.5 h and 2.5 h, except for a doubling of acid phosphatase in the soluble fraction, indicating increased lysosomal fragility (large pinolysosomes) as a result of the incubation. This is demonstrated more directly in Table 2, where the latency of acid phosphatase can be seen to be lower at 2.5 h than at 0.5 h. It should be noted that differences in latency arise during the enzyme assay as a result of lysosomal rupture; the initial latency is very high at both time points [4]. The data of Table 2 are thus consistent with the presence of largely intact lysosomes in all fractions except the very small particles (lysosomal membrane remnants?) and the soluble fraction. The amount of cell-corpse-associated [ C]sucrose increased 2.7-fold from 0.5 h to 2.5 h, the increase being almost exclusively in the larger-particle fractions. There was no increase in the very-small-particle fraction (which corresponds to a traditional microsome fraction), i.e. the endoplasmic reticulum apparently does not participate in [ 4C]sucrose sequestration. The radioactivity increase in the soluble fraction (1.4-fold) was even less than the increase in acid phosphatase (2.2-fold) despite the 2.7-fold increase in total cell-corpse-associated radioactivity, again pointing out that the sequestered [ C]sucrose resides in structures which are (on average) much less fragile than lysosomes. It should be kept in mind that the subcellular fractions in these experiments are not to be compared with the fractions traditionally prepared [l I], because the cytosol has already been removed prior to homogenization (cf. the low protein content of the soluble fraction). Table 1 makes it clear that acid phosphatase and sequestered [ C]sucrose have quite different particle size distributions. The largest particles contain relatively more

4 226 TOP I06 I00 I I BOTTOM DENSITY (glmll Fig. 2. Distribution of /'4C].suc.ra.~e-.saquestering organelles in metrizuniick grudimts. A cell corpse homogenatc, prepared from hcpatocytes which had been allowed to sequester ['4C]sucrose for 2 h at 37'C, was centrifuged on a metrizamidc/sucrose gradient at (L FRACTION NUM0ER 3.4 x lo6 g. min. Fractions were assayed for (0)['4C]sucrose; (0) hexosaminidase, and (a) cytochrome oxidase. In this and subsequent Fig. 3. Gel.jiltrution of rudiouctivit~ sequestered bj, Ijso.wniul[ mid gradient experimcnts (Figs 4-7), two or three top fractions and two mitochondriul.fructions. Peak fractions from the lysosomal (1.I0 g bottom fractions have been pooled. Values are expresscd as dpm/ ml) and mitochondria1 (1.I6 g/ml) peaks of metrizamide gradients of fraction or as percentages of the total in thc gr a d' lent the type shown in Fig. 2 were collected, and analysed on Scphadcx G-15 columns. Column fractions werc takcn for determination of ''C radioactivity, and the elution pattern was compared with dirferent markcrs. (A) Markers: (0) 1'4C]sucrose; (0) ['IiJgIucose; (0) blue ['4C]sucrose, while the smaller particlcs contain relatively more acid phosphatase (cf. the ratios given in the last column Of Table 1). This distribution pattern is unlikely to reflect just a preferential accumulation of [ ''C]sucrose in large lysosomes : large lysosomes are generally more fragile than small lysosomes [I31 (cf. also the medium-sized and small particles in Table 2), whereas the ['4C]sucrose-containing particles are less fragile. It would therefore, seem reasonable to conclude that at least part of the [14C]sucrose is sequestered in nonlysosoinal organelles. Isotonic metrizamide gradients separate subcellular components according to their native density, and have been found particularly useful in segregating lysosomes and mitochondria [7-91. When cell corpses containing sequestered ['4C]sucrose were homogenized and fractionated on such gradients, two distinct peaks of radioactivity were observed (Fig. 2), as reported previously [5]. The dense peak (1.15 g/ml) coincided with the mitochondrial marker enzyme, cytochrome oxidase, whereas the light peak ( g/ml) coincided with lysosomal marker enzymes, such as hexosaminidase (Fig. 2) and acid phosphatase (Fig. 4A). In addition, a variable amount (depending on the extent of homogenization-induced lysosomal damage) of non-sedimentable ['4C]sucrose was found in the top region of the gradient. This radioactivity includes, as a major contributor, a constant 'background' of soluble ['4C]sucrose (cf. Fig. 1) as well as the [14C]sucrose leaked from lysosomes. The bottom fractions, which include peroxisomes 1151 and pelleted nuclei, contained small and variable quantities of enzymes and [14C]sucrose. The actual physical association of the radioactivity peaks with mitochondria and lysosomes, respectively, has been verified by the use of agents which alter organelle density: the dense peak codistributes with mitochondria made denser with dextran (void-volume marker). (B) I4C radioactivity in suhccllular fractions: (0) lysosomal fraction; (e) mitochondrial fraction a nitrotetrazolium dye, whereas the light peak codistributes with lysosomes made lighter by means of the lysosomotropic drug chloroquine (51. It is, therefore, reasonable to assume that the observed disparity in sedimentability and subcellular particle size distribution between acid phosphatase and sequestered radioactivity in large measure can be ascribed to the presence of a considerable amount of ['4C]sucrose in mitochondria (E 1/3 of the total sequestered). How the radio- Distribution of (14C],~~~ro~~e-~~eq~~~~te~ing organellt~s in nzetrizamide density gradients activity in the light peak is distributed between lysosomes and autophagosomes is not precisely known, but the densityalteration experiments [5] suggest that most of it is lysosomal. Authenticity of [I C]sucrose in suhcellulur j k t ions To verify that the radioactivity in the gradient-separated organelles was authentic ['4C]sucrose and not some metabolic product, fractions from both the light and the dense peak were frozen/thawed and subjected to gel filtration on Sephadex G-I 5 columns (Fig. 3). With both fractions (Fig. 3B) the radioactivity eluted precisely in the position of the ['4C]sucrose marker (Fig. 3A), showing no evidence of metabolism to either smaller or bigger molecules (cf. the positions of [3H]glucose and dextran in Fig. 3A). Correlation between size und density of [I 4C/sucro.s~~-.~equestering organelles Mitochondria and lysosomes can be partially separated on the basis of size, using differential centrifugation (1 I]. A purified preparation of 'large mitochondria', i.e. a postnuclear, g. min pellet, was subjected to metrizamide gradient centrifugation. In the experiment shown in Fig. 4 this pellet fraction was essentially devoid of lysosomes (acid

5 221 A t 2a min 0 PELLET min C SUPERNATANT DENSITY IglmlI Fig. 5. Efject oj digitonin extraction on grudient distribution of seqrrestered [''C].rtrcro.re. Hepaiocytes were electropermcabilized. loaded with ['4C]sucrose and incubated for 2.5 h at 37 'C. One-half of the cells were electrodisrupted and used for preparation of cell corpse homogenates as usual; the other half was trcated with 0.1 % digilonin (instead of electrodisruplion) before cell corpse isolation/ homogenization. Thc homogenates were analyscd on metrizamide density gradients. (0) Control (electrodisrupted); (0) digitoninextracted 5 TOP 108 I10 I12 I I BOTTC DENSITY IgJmlI Fig. 4. Grudiirnt distribution of' sequestered [ L4C]sucro,se in different suhceilulir jiaction.r. Cell corpse homogenates, prepared from hepatocytes which had been electropermeabiliaed, loaded with [14C]st~crose and incubated for 2 h at 37 'C, were used for subccllular fractionation by differential centrifugation. After removal of nuclei, a lysosome-depleted fraction (i.e. purified mitochondria) was prepared by pelleting twice at x g. min. This fraction, as well as the rcmaining (combined) supernatant and a sample of the unfractionated homogenate, was analysed on metrizamide density gradients. (A) Total homogenatc; (B) lysosome-depleted (mitochondrial) pellet; (C) remaining supernatant (still containing about one-half of the mitochondria). (0) Sequcstered ['4C]sucrose; (0) acid phosphatase; protein (a) phosphatase) and of light peak radioactivity, but contained about one-half of the [14C]sucrose sequestered in the dense peak (Fig. 4B). The remaining supernatant (Fig. 4C) contained the rest of the dense-peak radioactivity plus all of the light-peak radioactivity present in the original homogenate (Fig. 4A). Such supernatants are known to contain both lysosomes and small mitochondria [I I]; cf. the distribution patterns of protein and acid phosphatase in Fig. 4C. The data thus provide relatively clear-cut support of the notion that the dense peak represents mitochondrial [14C]sucrose and the light peak lysosomal [14C]sucrose. Digitonin extruction of light-peak radiouctivity Digitonin is a selective detergent which, at low concentrations, may dissolve most cellular membranes while leaving the mitochondria intact [16]. As shown in Fig. 5, treatment or hepatocytes with digitonin before homogenization effectively extracted the ['4C]sucrose sequestered in the light peak, while leaving the dense-peak radioactivity unaffected. The virtually 0 in - (14C)SUCROSE rn N - DIGITONIN CONCENTRATION (rng/rnl) Fig. 6. Destruction qf mitochondriu at high, hut not at low), digitonin concentrations. Hepatocytic cell corpses were treated briefly with digitonin at the concentration indicated, then sedimcnted by centrifugation. Thc amount of extracted ['4C]sucrose and of the mitochondrial matrix enzyme, malate dehydrogenase, was measured in the supernatant. Each value is the mean of three parallel cell samples complete absence of radioactivity in the light region of the gradient suggests that both lysosomes and autophagosomes are digitonin-sensitive. Digitonin titration studies (P. 9. Gordon and P. 0. Seglen, unpublished results) have shown that 0.3 mg/ml is sufficient for complete extraction of lysosome-associated [14C]sucrose from hepatocytic cell corpses. Following a well-defined doseresponse plateau, extraction of mitochondrial ['4C]sucrose begins as digitonin concentrations approach 1 mg/ml (notice that digitonin is effective at lower concentrations when applied to cell corpses than when applied to whole cells). Fig. 6 shows that solubilization of a mitochondrial matrix enzyme, malate dehydrogenase, parallels the extraction of ['4C]sucrose at high digitonin concentrations, confirming the

6 when ['4C]sucrose was present both intracellularly and extracellularly. It should be noted that in addition to its effect on light-peak radioactivity, 3-methyladenine markedly retarded the time-dependent shift in lysosomal density, suggesting that the latter may be at least partially a result of autohagic activity ~71. In contrast, the dense (1.15 g/ml) ['4C]sucrose peak was not significantly affected by 3-methyladenine. Uptake into this peak would, therefore, appear to be unrelated to autophagic-lysosomal activity, as might be expected of a process associated with the mitochondria. The 3-methyladenine experiment thus confirms the clear-cut distinction between autophagic-lysosomal sequestration, represented by the light gradient peak, and mitochondrial uptake, represented by the dense peak. DENSITY IglrnlI Fig. 7. Effect oj3-methyladenine and incubation time on gradient di.rtributions of acid phosphatase and sequestered [''C]sucrose. Electroperineabilized hepatocytes were loaded with ['4C]sucrose (1 h at O'T) and preincubated for 30 min at 37 C (A), then incubated for another 2 h in the absence (B) or presence (C) of 3-methyladenine (10 mm). Cell corpse homogenates were analysed on metrizamide density gradients. (0)['4C]Sucrosc; (0) acid phosphatase. (......) The distribution of cytochrome oxidase (from a different experiment) supposedly mitochondrial location of the latter. At 0.3 mg/ ml, on the other hand, all of the radioactivity ascribed to lysosomes (z 50% of total in the experiment shown in Fig. 6) is extracted, with only a 5% loss of the mitochondrial marker enzyme (data omitted from Fig. 6 for a clearer display of mitochondria solubilization). Digitonin, at the appropriate ceoncentration, can thus be used to distinguish rather sharply between lysosomai and mitochondria1 localization of soluble compounds. EfJict of 3-methyladenine on density distributions of acid phosphatase and sequestered ('4C]sucrose The autophagy inhibitor 3-methyladenine strongly suppresses ['4C]sucrose sequestration in hepatocytes [4], and Fig. 7 shows that the drug selectively affects sequestration into the light (autophagosomal/lysosomal) density-gradient peak. Whereas a prominent light (1.08 g/ml) ['4C]sucrose peak is seen in the control at 150 min (Fig. 7 B), the radioactivity in the 3-methyladenine-treated cells (Fig. 7C) is only slightly above the background (Fig. 7A) in this region. The gradient distribution of cytochrome oxidase is included in Fig. 7 C to indicate that some cross-contamination by labelled mitochondria may extend into the light-peak region and account in part for the slight rise above background. The background radioactivity in the light region may furthermore include some pinosomes hbelled during the 30-min period DISCUSSION The gradient experiments show that intracellular sequestration of electroinjected ['4C]sucrose can be resolved into two distinct processes. (a) Autophagic-lysosomal sequestration is the most likely mechanism for [14C]sucrose accumulation in the light ( g/ml) density gradient peak. The radioactivity in this region coincides (within 1-2 h of incubation) with lysosomal marker enzymes, and can be shifted to lower densities upon loading of the lysosomes with an acidotrophic amine [5]. The radioactivity resides (at least for the major part) in digitonin-sensitive vesicles, which are larger than microsomes but smaller than large mitochondria. These characteristics are consistent with a predominant localization in lysosomes, which would bc the expected end destination of autophagically sequestered sucrose. It should be pointed out, however, that during continuous autophagy a certain steadystate level of [14C]sucrose will necessarily have to be maintained in the initial sequestrating compartment. the autophagosomes, a fact which should not be set aside by our present inability to distinguish between autophagosomes and lysosomes. The strong and selective suppression of the light ['4C]sucrose peak by 3-methyladenine, a specific inhibitor of autophagic-lysosomal protein degradation [18], testifies to autophagy as the origin of the radioactivity in this peak. The bioregulation by 3-methyladenine and amino acids as well as the strong temperature-sensitivity of the lysosomal ['4C]sucrose accumulation (P. B. Gordon and P. 0. Seglen. unpublished results) would furthermore seem to rule out diffusion into the lysosome as a significant contributory process. It has been suggested that autophagic sequestration may take place both through the formation of autophagosomes (which subsequently fuse with lysosomes) and by a direct invagination of the lysosomal membrane (microautophagy) [19]. The present gradient experiments do not specifically address this issue. Net ['4C]sucrose accumulation is detectable in the light-peak region before the (initially dense) acid phosphatase peak has become coincident with the radioactivity peak [17]; however, the number of lysosomes in this region is sufficient to mask any distinction between lysosomes and autophagosomes, or between (macro)autophagy and microautophagy. In the denser region of the gradient the mitochondrial ['4C]sucrose uptake is too dominant to permit any assessment of microautophagy into dense lysosomes. (b) Mitochondria1 ['4C]sucrose uptake would seem to be responsible for the accumulation of radioactivity in the dense (1.15 g/ml) gradient peak. This peak coincides with a mitochondrial marker enzyme as well as with the major protein

7 229 peak, and shifts its position upon loading of mitochondria with a dense oxidation-substrate [5]. The dense [ ''C]sucrose peak has sedimentation characteristics similar to mitochondria, and is present in purified (essentially lysosome-free) heavy mitochondrial fractions. The peak radioactivity exhibits the characteristic mitochondrial resistance towards digitonin treatment [I61 and is unaffected by 3-methyladenine at concentrations up to 10 mm. (At higher 3-methyladenine concentrations, progressive inhibition of mitochondrial ['4C]sucrose uptake is demonstrable, accounting for the greater effect of 3-methyladenine than of amino acids on overall ['4C]sucrose sequestration [41.) Since the mitochondrial ['4C]sucrose uptake is unaffected by 3-methyladenine as well as by amino acids (results not shown), it is clearly unrelated to autophagic-lysosomal sequestration. (To avoid confusion, we prefer to reserve the term 'sequestration' for the autophagic process or for the overall transfer of ['4C]sucrose to sedimentable structures, and rather use the word 'uptake' when dealing specifically with the mitochondrial process.) We have furthermore found that mitochondria are also able to take up the disaccharide lactose, but not the trisaccharide raffnose (results not shown); the uptake is, therefore, apparently limited to molecules of small molecular mass and consequently unlikely to relate to any form of macromolecular degradation, including mitochondrial protein degradation [20]. We have not attempted to reproduce the in situ mitochondrial ['4C]sucrose uptake (i.e. that occurring in intact hepatocytes) by using isolated mitochondria. It is well established that isolated mitochondria are impermeable to, and do not take up sucrose [21, 221, and it is possible that the in situ uptake may depend, for example, on intact connections between mitochondria and the endoplasmic reticulum [23]. Our finding that the mitochondrial [14C]sucrose uptake is both highly temperature-sensitive and energy-dependent (P. B. Gordon and P. 0. Seglen, unpublished results) would argue for a process more complicated than a simple diffusion. However, the assignment of a defined functional role for mitochondrial disaccharide uptake will have to await further investigation. This project is generously supported by The Norwegian Cancer Society. REFERENCES 1. Stacey, D. W. & Allfrey, V. G. (1977) J. Cell Bid. 75, Zavortink, M., Thacher, T. & Rechsteiner, M. (1979) J. Cell Physiol. 100, Gordon, P. B. & Seglen, P. 0. (1982) Exp. Cell Res. 142, Seglen, P. 0. & Gordon, P. B. (1984) J. Cell Bid. 99, Tolleshaug, H., Gordon, P. B., Solheim, A. E. & Seglen, P. 0. (1984) Biochem. Biophys. Res. Commun. II9> Tolleshaug, H. (1981) Int. J. Biochem. 13, Schwarze, P. E., Tolleshaug, H. & Seglen, P. 0. (1985) Carcinogenesis 6, Seglen, P. 0. (1976) Methods Cell Bid. 13, Seglen, P. O., Gordon, P. B. & Poli, A. (1980) Biochim. Biophys. Acta 630, Solheim, A. E. & Seglen, P. 0. (1980) Eur. J. Biochem Duve, C. de, Pressman, B. C., Gianetto, R., Wattiaux, R. & Appelmans, F. (1955) Biochem. J. 60, Barrett, A. J. (1972) in Lysosomes, a laboratory handbook (Dingle, J. E., ed.) pp , North-Holland, Amsterdam. 13. Solheim, A. E. & Seglen, P. 0. (1983) Biochim. Biophys. Acta 763, Cooperstein, S. J. & Lazarow, A. (1951) J. Bid. Chern. 189, Wattiaux, R., Wattiaux-de Coninck, S., Ronveaux-Dupal, M.- F. & Dubois, F. (1978) J. Cell Bid. 78, Zuurendonk, P. F. & Tager, J. M. (1974) Biochzm. 5iophj.s. Acta 333, Seglen, P. 0. & Solheim, A. E. (1985) Exp. Cell Res. 157, Seglen, P. 0. & Gordon, P. B. (1982) Proc. Nut1 Acad. Sci. USA 79, Glaumann, H., Ericsson, J. L. E. & Marzella, L. (1981) Int. Rev. C.vtol. 73, Desautels, M. & Goldberg, A. L. (1982) Proc. Nut1 Acud. Sci. USA 79, Halestrap, A. P. & Quinlan, P. T. (1983) Biochem. J. 214, Quinlan, P. T., Thomas, A. P., Armston, A. E. & Halestrap, A. P. (1983) Biochem. J. 214, Katz, J., Wals, P. A., Golden, S. & Raijman, L. (1983) Biochem. J. 214,