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Separation of endocytic vesicles in Nycodenz gradients
ANALYTICAL
BIOCHEMKTRY
Separation
142,455-462
(1984)
of Endocytic Vesicles in Nycodenz Gradients
GRETE M. KINDBERG, DAVID
TERENCE FORD,* RUNE BLOMHOFF,
RICKWOOD,*
AND TROND
BERG
Institute for Nutrition Research, University of Oslo, P.O. Box 1046, Blindem, *Department of Biology, University of Essex, Wivenhoe Park, Colchester,
0316 Oslo 3, Norway, Essex, United Kingdom
and
Received February 7, 1984 The endocytosis of ‘251-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/ sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after l/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligandcontaining endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies. o 1984 Academic PW, IX.
Heterophagy of asialoglycoproteins in hepatocytes is a multistep process. After binding to receptors located in coated pits, the ligand is internalized into coated vesicles (1). The receptor-ligand complex is subsequently found in smooth-surfaced vesicles. Acidification of these vesicles (2) leads to dissociation of ligand and receptor, and these two components are sequestered into different organelles which eventually mediate the transport of the receptor back to the plasma membrane and the ligand to the lysosomes (3). The intracellular transport of labeled asialoglycoproteins in hepatocytes has been studied by electron microscopy (4-7) and subcellular fractionation techniques (8- 10). Sucrose gradient centrifugation in combination with differential centrifugation has indicated that the asialoglycoprotein after uptake into the cells is in vesicles which increase in size and density (9-12). Eventually the ligand enters the lysosome. By using asialofetuin derivatized with 14C-sucrose, the labeled degradation products are trapped in
the lysosome (13,14), and it can be shown that the distribution of labeled degradation products in a gradient coincide with the distribution of lysosomal enzymes. Relatively good separations of endocytic vesicles (containing labeled asialoglycoprotein) and lysosomes can also be obtained using Percoll gradients (2,15). The fractionation techniques employed so far have not identified the early steps in the uptake process, although morphological data suggest that the asialoglycoprotein is transferred from a relatively small vesicle to larger tubular structures (5,s). The purpose of the present study was to examine the subcellular distribution of rz51-labeled asialofetuin (AF)’ early after its uptake into isolated rat hepatocytes. Fractionation of isolated rat hepatocytes was carried out using isotonic Nycodenz/sucrose density gradients. The data ob’ Abbreviations used: AF, asialofetuin; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 455
0003-2697184
$3.00
Copyright 0 1984 by Academic press. Inc. All rights of reproduction in any form reserwd
456
KINDBERG
tamed suggest that the ligand passes through two distinct vesicular structures on its way to the lysosomes. MATERIALS
AND
METHODS
Materials. Nycodenz and Maxidens were obtained from Nyegaard and Company (Oslo, Norway). ‘251-Labeled asialofetuin was prepared as described elsewhere ( 16). Collagenase and bovine serum albumin (BSA) were obtained from Sigma Chemical Company (Poole, Dorset, U.K.) All other chemicals were of the purest grades available. Preparation of puriJied hepatocytes. Isolated rat liver cells were prepared from male Wistar rats (200-250 g) by collagenase perfusion (17,18). The total liver cell suspension was incubated for 30 min at 37”C, sieved through nylon mesh, and centrifuged for 30 s at 5Og to pellet the hepatocytes. The pellet was resuspended in ice-cold minimal salt solution containing 1% (w/v) BSA (9) and recentrifuged twice. The resulting cell suspension of almost pure hepatocytes was 90- 100% viable as judged by the trypan blue exclusion test. Binding and internalization of the ligand. The hepatocyte suspension was incubated for 60 min at 4°C in the presence of 1251-labeled asialofetuin (2 &ml) to allow the ligand to bind to cell surface receptors. The ligand is not internalized at this temperature (16). The cells were washed three times in the minimal salt solution with 1% BSA at ice-cold temperature to remove unbound l&and. Cell concentration at this time was 5- 10 X lo6 cells/ml. Aliquots of cell suspension were incubated at 37°C for various time periods to allow internalization of the ligand. Warm incubation medium was added to the icecold aliquot to start the incubation and at the end of the required time period ice-cold medium was added and the mixture kept in ice. After incubation each aliquot was washed in ice-cold 0.25 M sucrose and homogenized in an ice-cold Dounce homogenizer with a tight-fitting pestle (9,lO). The homogenate was centrifuged for 115 s at 2000g in a
ET
AL.
Sorvall SS24 rotor, the supematant was kept, and the pellet was rehomogenized in 0.25 M sucrose. After centrifugation the second supematant was combined with the first. Centrifugation conditions. Preformed gradients were made up in 38-ml centrifuge tubes using a two-chamber mixer. The gradients were formed with 0.25 M sucrose solution containing 1 mM EDTA and 1 mM Hepes (pH 7.2) and a 45% (w/v) Nycodenz solution also containing 1 mM EDTA and 1 mM Hepes (pH 7.2). The resulting gradients had the density range 1.04-l .20 g/ml. The cytoplasmic extract was top loaded onto the gradients (4 ml/gradient) and centrifuged at 4°C at 85,OOOg for the times shown in the text and figures. Each gradient was fmctionated by upward displacement, using Maxidens as the displacement fluid, into 2.0-ml fractions. The density of each fraction was determined using the equation: density = 3.4 In - 3.55, where n is the refractive index at 20°C. This is the equation given for isotonic Nycodenz/sucrose gradients (19) and, in the case of the gradients used here, gives a useful approximation of the density of fractions. ’ The distribution of the ligand was determined by use of a gamma counter, membrane distribution by 5’nucleotidase assay (20), protein by Coomassie blue assay (21), lysosomes by acid phosphatase assay ( lo), and peroxisomes by catalase assay (22). RESULTS
Sedimentation Properties of Newly-Formed Endocytic Vesicles Morphological studies have indicated that asialoglycoproteins during their intracellular transport in hepatocytes are found in vesicles of increasing size (5,8). This change in size is likely to be reflected in variations in their rate of sedimentation in Nycodenz gradients. To determine the centrifugation time needed to get the newly formed (and smallest) endocytic vesicles to their isopycnic distribution in the gradients, cytoplasmic extracts were prepared 1 min after the start of the uptake
SEPARATION
OF ENJXXYTIC
of ‘251-labeled AF at 37°C. The cells had been previously incubated for 60 min with labeled ligand at 4°C as described under Materials and Methods. In preliminary experiments the density distribution of ‘251-labeled AF and various marker enzymes in gradients was determined following application of the cell homogenate on top and at the bottom of the gradient. In the experiment depicted in Fig. 1, the cytoplasmic extracts were prepared after incubating the cells for 1 min at 37°C. The tubes were centrifuged for 1 h at 85,000g. The results showed that the marker enzymes for lysosomes (acid phosphatase), mitochondria (cytochrome ox&se), peroxisomes (catalase), and the plasma membrane (5’nucleotidase) were practically identical for top-loaded and bottom-loaded gradients. Therefore, these organelles had reached their equilibrium density in the gradient. On the other hand, the distributions of rz51-labeled AF did not coincide, indicating that the newly formed endocytic vesicles were not distributed isopycnically. The cells were washed in a buffer
“1.05
1.10 Density
1.15 (g/ml)
FIG. 1. Distribution patterns of *251-labeled AF in Nycodenz gradients after loading postnuclear fractions on top (filled symbols) or at the bottom (open symbols) of the gradients. The postnuclear fraction was prepared from cells that had been preincubated at 4°C for 60 min in the presence of 1251-labeledAF (1 nM) and then, after washing, incubated for I min at 37°C. The tubes were centrifuged for 1 h (circles) or 2 h (squares) at 85,000g. The radioactivities in the gradients are plotted against the density of the fractions. Only radioactivity in the gradient itself is shown.
VESICLES
457
-I
1 2 1.05 Fr. nr.
1.10
1.15
Density
FIG. 2. The effect of centrifugation time on the distribution of 12sI-labeledAF in Nycodenz gradients. Aliquots of postnuclear fractions were prepared from cells that had been given a 4”C-binding period of labeled AF and then incubated at 37°C for 1 min. The postnuclear fractions were layered on top of the gradients and centrifuged at 85,000g for 1 (O), 2 (A), or 3 h (0). Radioactivities were determined in the fractions and are plotted against density of the fractions, except for the two upper fractions which contain material not entering the gradient itself.
containing 4 mM EDTA before homogenization. Hence, only internalized ligand is recovered in the gradients (16). To determine the time needed for the newly formed endocytic vesicles to reach their bouyant density during centrifugation at 85,OOOg, homogenates were again prepared from cells that had been incubated at 37°C for 1 min (after a binding period of ‘251labeled AF at 4°C). The results showed that the distribution of radioactivity migrated further down the gradient with increasing centrifugation times up to 3 h. Figure 2 shows the distribution of ‘251-labeled AF in gradients centrifuged for 1, 2, and 3 h at 85,000g. The cytoplasmic extracts prepared 1 min after the start of the uptake of ‘251-labeled AF were layered on top of the gradients. Increasing the time of centrifugation up to 4 h produced no further migration of the vesicles, indicating that they had reached their isopycnic position after 3 h of centrifugation at 85,000g. Peak radioactivity was then at a density of 1.11 g/ml.
458
KINDBERG
Separation of Endocytic Vesicles Based on Differences in Sedimentation Rate The results in Fig. 2 show that the newly formed endocytic vesicles containing AF slowly move toward their isopycnic position. If the endocytic vesicles subsequently increase in size, such size differences should allow the separation of vesicles at various stages to be carried out. In the experiment depicted in Fig. 3 the cytoplasmic extracts were prepared from cells that were preincubated with ‘251-labeled AF at 4°C and then, after washing, were incubated at 37°C for 30 set, and 1, 2.5, 15, and 30 min. The cytoplasmic extracts were layered on top of the gradients and centrifuged for 45 min at 85,OOOg. The distribution of the ligand (Fig. 3) shows clearly the differences in the sedimentation behavior of the particles after different incubation times. After 30 s the small amount of internalized ligand was all in the form of slowly sedimenting particles. After 1.0 min, the amount of internalized ligand was more than doubled, consisting of a prominent peak of “slow”-sedimenting vesicles and populations of faster-moving particles. After incubation for 2.5 min slow-moving vesicles were still apparent, but much larger populations 0
,
Fr. nr.
1
Density
FIG. 3. Distribution of ‘251-labeled AF containing endocytic vesicles in Nycodenz gradients after short (45 min) centrifugation time. Following incubation with “rIlabeled AF at 4°C for 60 min cells were washed and then incubated at 37°C for 0.5 (O), 1 (A), 2.5 (A), 15 (m), and 30 min (0). Postnuclear fractions were subsequently prepared and layered on top of linear Nycodenz gradients. The tubes were centrifuged for 45 min at 85,OOOg.The results are presented as described in Fig. 2
ET AL.
of the faster-moving vesicles had developed. After 15 min, all the internalized ligand is incorporated in the fast-sedimenting vesicles, as is the reduced amount of ligand present after 30 min of incubation. The results demonstrate that, initially, the vesicles are small and of similar size. With increasing incubation times, more ligand is endocytosed and the vesicles, in subsequent steps, increase in size and, thus, sediment at a faster rate. Recent electron microscopic investigations on the fractions obtained after 45 min of centrifugation in Nycodenz gradients confirm the differences in size between vesicles in fractions with densities of about 1.05 and fractions with densities of about 1.10 g/ml (T. Ford, data to be published). The amount of ligand recovered in the gradients was, as expected, dependent on the time of incubation of the cells. It is known that during incubation of 4°C the ligand is bound to cell surface receptors but not internalized and, when the cells are washed and incubated at 37°C the surface-bound ligand is internalized with a half-time of about 3 min (16). After 15 min all ligand is intemalized. At approximately this time point the AF starts to be degraded (23), and as the degradation products leave the lysosomes as well as the cells very quickly (23,24), the cellassociated radioactivity starts to decrease. Although the amount of cell-associated radioactivity is fairly constant during the first 15 min of incubation, the amount of radioactivity recovered in the gradients increases with incubation time up to 15 min. This is due to the fact that only internalized ligand enters the gradients. Ligand associated with surface receptors is released from the cells when they are washed in calcium-free sucrose solutions before homogenization. Isopycnic Distributions of ‘“I-Labeled AF at Various Times after the Start of the Internalization When cytoplasmic extracts, prepared after various periods of incubation to internalize
SEPARATION
OF ENDOCYTIC
A F:06 7
VESICLES
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FIG. 4 (A) Distribution of ‘2sI-labeled AF after isopycnic centrifugation in Nycodenz gradients of postnuclear fractions. The fractions were prepared from cells that were incubated at 37°C for 1 (A), 2.5 (O), and 15 min (0) following preincubation at 4°C with labeled AF. The centrifugation was carried out for 3 h at 85,000g. Results are presented as described in Fig. 2. (B) Distributions of enzymes after isopycnic centrifugation in a Nycodenz gradient of a postnuclear fraction. The density distributions of catalase (A), 5’-nucleotidase (0), and acid phosphatase (0) are shown. The enzyme activities in the fractions are presented as percentages of total recovered activity in the tube and are plotted against the density of the fraction, excepting the two top fractions which contain material not entering the gradient
the ligand, were centrifuged at 85,000g for 3 h it was found that the density distribution was very similar for vesicles containing newly internalized ligand and those prepared up to 30 min after the start of the internalization of the ligand. Figure 4 depicts the density distributions of ligand that had been internalized into the cells for 1, 2.5, and 15 min. Peak activity is in all cases at about 1.11 g/ ml. Included in the figure are distribution curves for lysosomes (acid phosphatase), plasma membrane (5’-nucleotidase), and peroxisomes (catalase). Do the Endocytic Vesicles Change in Size and/or Density during Centrifugation in Nycodenz Gradients? The possibility that AF-containing vesicles change in size and/or density during centrifugation in Nycodenz gradients was considered. To get information about this, the following experiment was done: Cells were first allowed to internalize ‘251-labeled AF for 1 min at 37°C following the l-h binding period at 4°C. Postnuclear fractions were prepared and centrifuged in Nycodenz gradients for either 45 min or 3 h at 85,000g.
The results were as expected (Fig. 5). After a 45-min centrifugation peak radioactivity was in the gradient at a density of 1.06 g/ml; and after 3 h of centrifugation the peak was at 1.11 g/ml. Fractions 10 and 11 (counted from the top of the gradient) in the gradient centrifuged for 3 h were pooled and diluted with 20 vol of 0.25 M sucrose, and samples of this preparation were then recentrifuged for 45 min in new Nycodenz gradients. The results are depicted in Fig. 6, and show that the vesicles had retained their sedimentation properties fairly well during the centrifugation at 85,000g for 3 h. The peak in the distribution curve is still at about 1.06 g/ml. Efects of Ammonia on the Density Distribution of 12’1-Labeled AF in Nycodenz Gradients Cells were first allowed to bind ligand at 4°C. They were then washed and reincubated at 37°C in the presence or absence of 10 mM NH&l. At various time points after the start of the incubation at 37°C cytoplasmic extracts were prepared and centrifuged for 45 min at 85,000g at 4°C. The results show (Fig. 6) that the presence of NH; does effect
460
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the curves generated by the vesicles after 60 min incubation in the presence or absence of the inhibitor shows that degradation is much slower in the presence of the inhibitor. Our results suggest that the presence of NH: reduces the rate of degradation by acting upon the maturation of the endosomes; the ligand must be located in the fastersedimenting form to be available to the lysosomes. During incubation of the cells at 37°C in the presence or absence of NH:, a reduction in total recovered radioactivity in the gradient could be completely accounted for by a corresponding increase in acid-soluble radioactive degradation products (not shown).
Distribution of Free and Bound Ligand in the Cells
115
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ET AL.
glml
FIG. 5. Effects of centrifugation in Nycodenz gradients on the sedimentation properties of newly formed % labeled AF-containing vesicles. Hepatocytes were incubated for 1 min at 37°C following a l-h binding period at 4’C in the presence of ‘251-labeled AF. Postnuclear fractions were prepared and centrifuged in Nycodenz gradients for 45 min (A) or 3 h (B) at 85,000g. Following fractionation, fractions 10 and 1 I (densities 1.09 and 1.10 g/ml) in the gradient centrifuged for 3 h (B) were pooled and diluted with 20 vol of 0.25 M sucrose. This preparation was recentrifugcd at 85,000g for 45 min (C). The radioactivity in the fractions is plotted against the density of the fractions and is presented as the percentage of total recovered radioactivity in the gradient.
Fig. 7 shows the distribution of free and receptor-bound ligand in the cells 1, 5, and 15 min after the start of the incubation at 37°C. These data, which were obtained by separating free and receptor-bound ligand in a G- 100 column, show that almost all ligand is bound in the “small” vesicle while an increasing fraction of ligand is free in the “large” vesicle. However, even after 15 min,
J
Fr. nr.
the distribution of the ligand. A large proportion of the ligand is retained in the form of slowly sedimenting particles, although the larger, faster-sedimenting species are still present in reduced amounts. Degradation of the ligand still occurs, judging by the reduction of the total 12’1 present with increasing incubation time. However, comparison of
Density
FIG. 6. Effect of NH; on the distribution of lz51lab&xl AF in Nycodenz gradients after a short (45-min) centrifugation. Cells were. first incubated at 4°C for 60 min in the presence of the labeled ligand. They were then washed and incubated at 37°C in the presence (open symbols) or absence (filled symbols) of 10 mM NH:. Postnuclear fractions were prepared after 15 and 30 min and layered on top of Nycodenz gradients, which were subsequently centrifuged for 45 min at 85,000g. The results are presented as described in Fii. 2.
SEPARATION
OF ENDGCYTIC
VESICLES
461
receptor dissociated. A similar intracellular transport of asialoglycoproteins was proposed by Hubbard et al. (5-7). The fractionation method described in this report makes it possible to precisely analyze the kinetic aspects of the intracellular transport of endocytosed ligand. The method should therefore be particularly useful for evaluating the effects of drugs and hormones on the heterophagic process. Studies involving gel filtration showed that most of the ligand was bound in the “early” small vesicle while increasing amounts became free in the larger vesicle. These data are compatible with the finding that pH is Fraction nr. lowered in the endocytic vesicle (15,25) and FIG. 7. Gel filtration in Sephadex G-100 of solubilized that the affinity of receptor for the asialoglycells containing ‘2’I-labeled AF. The cells were first coprotein decreases with decreasing pH (2). incubated at 4°C for 60 min with ‘*“I-labeled AF. They were then washed and reincubated at 37°C for 1, 5, and The results are in agreement with the mor15 min. The cells were chilled and treated with 1% phological data presented by Geuze et al. (8). They found that the ligand was associated Triton X-100 in the presence of an excess of unlabeled AF (to prevent reassociation of ligand to the receptor) with the membrane of the initial small vesicle (4). Bound and free ligand were separted by gel filtration and that it was free in the lumen of the through a Sephadex G-100 column (0.9 X 30 cm): The “CURL” (compartment of uncoupling of column was eluted with a minimal salt solution containing receptor and ligand). Harford et al. (3), using Hepes (pH 7.4). The position of free 12SI-labeled AF after a technique similar to ours, also observed gel filtration (indicated by an arrow) was determined by adding the labeled ligand to solubilized cells and then that the ligand became free in the endocytic running the preparation through the column. We have vesicle. shown previously that the receptor-ligand complex In the presence of ammonia and monensin emerges from the column close to the void volume (26). the ligand seemed to be retained in the “early” vesicle. Both these compounds will about 30% of the ligand is still associated prevent the drop in pH which normally takes with the receptor. place in endocytic vesicles (15,25). It is possible that the acidification of the small enDISCUSSION docytic vesicles is necessary for their develThe results obtained in this study suggest opment (by fusion) into the larger endocytic that lz51-labeled AF internalized into hepa- vesicle. tocytes is transported to the lysosomes ACKNOWLEDGMENTS through at least two types of vesicular strucThe authors are grateful to Ms. Kari Holte for expert tures. Initially the ligand is in a relatively small vesicle, but after 5 min its transfor- technical assistance and to Ms. Ba Gsrbitz for preparing the manuscript. The work was financially supported by mation to larger vesicles is almost complete. the Norwegian Council for Science and the Humanities, The two-step process suggested by the the Nansen Foundation, and the Norwegian Council on present data is in agreement with earlier Cardiovascular Diseases. reports: Geuze et al. (8) showed that asialoREFERENCES glycoproteins were transferred from small vesicles located at the surface of the cell to 1. Ashwell, G., and Harford, J. (1982) Annu. Rev. larger vesicles in which the ligand and the Biochem. 51, 53 l-554.
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2. Harford, J., and Ashwell, G. (1982) in The Glycoconjugates (Horowitz, M. I., ed.), Vol. 4, pp. 2755, Academic Press, New York. 3. Harford, J., Wolkoff, A. W., Ashwell, G., and Klausner, R. D. (1983) J. Cell Biol. 96, 1824-1828. 4. Bridges, K., Harford, J., Ashwell, G., and Klausner, R. D. (1982) Proc. Natl. Acad. Sci. (USA) 79, 350-354.
Wall, D. A., Wilson, G., and Hubbard, A. L. (1980) Cell 21, 79-93. 6. Hubbard, A. L., Wilson, G., Ashwell, G., and Stukenbrok, H. (1979) J. Cell Biol. 83, 47-64. 7. Zeitlin, P. L., and Hubbard, A. L. (1982) J. Cell Biol. 92, 634-647. 8. Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F., and Schwartz, A. L. (1983) Cell 5.
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Tolleshaug, H., Berg, T., Frolich, W., and Norum, K. R. (1979) Biochim. Biophys. Acta 585, 7 l-84. IO. Berg, T., Ose, T., Ose, L., and Tolleshaug, H. (1981) Int. J. B&hem. 13, 253-259. 11. Tolleshaug, H., and Berg, T. (198 1) Exp. Cell Res.
ET AL. 14. Berg, T., Blomhoff, R., Naess, L., Tolleshaug, H., and Drevon, C. A. (1983) Exp. Cell Rex 148, 3 19-330.
15. Merion, M., and Sly, W. S. (1983) J. Cell Biol. 96, 644-650.
16. Tolleshaug, H. (1981) Znt. J. Biochem. 13, 45-51. 17. Seglen, P. 0. (1976) Methods Cell Biol. 13, 29-83. 18. Berg, T., and Blomhoff, R. (1983) in Iodinated Density Gradient Media (Rickwood, D., ed.), pp. 173-174, IRL Press, Oxford. 19. Rickwood, D. (1983) in Iodinated Density Gradient Media (Rickwood, D., ed.), pp. l-21, IRL Press, Oxford. 20. El-Aaser, A. A., and Reid, E. (1969) Histochem. J. 1,417-437.
21. Bradford, M. M. (1976) Anal. B&hem. 22.
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23. 24.
134, 207-217.
12. Tolleshaug, H., Biochim. Biophys. Acta, in press. 13. Pittman, R. C., Green, S. R., Attie, A. D., and Steinberg, D. (1979) J. Biol. Chem. 254, 68766879.
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Baudhuin, P., Beaufay, H., Rahman-Li, Y., Sehinger, 0. Z., Wattiaux, R., Jacques, P., and De Duve, C. (1964) Biochem. J. 92, 179-184. Tolleshaug, H., Berg, T., Nilsson, M., and Norum, K. R. (1977) B&him. Biophys. Acta 499,73-84. LaBadie, J. H., Peterson Chapman, K., and Aronson, N. N. (1975) B&hem. J. 152,271-279. Tyco, B., and Maxfield, F. R. (1982) Cell 28, 643651. Blomhoff, R., Tolleshaug, H., and Berg, T. (1982) J. Biol. Chem. 257. 7456-7459.
BIOCHEMKTRY
Separation
142,455-462
(1984)
of Endocytic Vesicles in Nycodenz Gradients
GRETE M. KINDBERG, DAVID
TERENCE FORD,* RUNE BLOMHOFF,
RICKWOOD,*
AND TROND
BERG
Institute for Nutrition Research, University of Oslo, P.O. Box 1046, Blindem, *Department of Biology, University of Essex, Wivenhoe Park, Colchester,
0316 Oslo 3, Norway, Essex, United Kingdom
and
Received February 7, 1984 The endocytosis of ‘251-labeled asialofetuin by rat hepatocytes was studied using Nycodenz/ sucrose gradients. It was shown in pulse chase experiments that the ligand endocytosed initially (after l/2 to 1 min) was in small, slow-sedimenting vesicles of similar sizes. The vesicles containing the ligand increased in size, and after about 2.5 min 20-30% of the ligand was recovered in larger, faster-sedimenting vesicles. After 15 min almost all internalized ligand was recovered in the fast-sedimenting vesicles. The initial, small endocytic vesicles and the later, larger endocytic vesicles have similar buoyant densities; the maturation of the endosomes can only be revealed by rate sedimentation, not by isopycnic centrifugation. Dissociation of ligand from receptor was found to occur in the larger, faster-sedimenting vesicles. The presence of ammonia inhibited the increase in size of the ligandcontaining endosomes. The methods employed here offer the possibility of obtaining endocytic vesicles at various stage of their development for further studies. o 1984 Academic PW, IX.
Heterophagy of asialoglycoproteins in hepatocytes is a multistep process. After binding to receptors located in coated pits, the ligand is internalized into coated vesicles (1). The receptor-ligand complex is subsequently found in smooth-surfaced vesicles. Acidification of these vesicles (2) leads to dissociation of ligand and receptor, and these two components are sequestered into different organelles which eventually mediate the transport of the receptor back to the plasma membrane and the ligand to the lysosomes (3). The intracellular transport of labeled asialoglycoproteins in hepatocytes has been studied by electron microscopy (4-7) and subcellular fractionation techniques (8- 10). Sucrose gradient centrifugation in combination with differential centrifugation has indicated that the asialoglycoprotein after uptake into the cells is in vesicles which increase in size and density (9-12). Eventually the ligand enters the lysosome. By using asialofetuin derivatized with 14C-sucrose, the labeled degradation products are trapped in
the lysosome (13,14), and it can be shown that the distribution of labeled degradation products in a gradient coincide with the distribution of lysosomal enzymes. Relatively good separations of endocytic vesicles (containing labeled asialoglycoprotein) and lysosomes can also be obtained using Percoll gradients (2,15). The fractionation techniques employed so far have not identified the early steps in the uptake process, although morphological data suggest that the asialoglycoprotein is transferred from a relatively small vesicle to larger tubular structures (5,s). The purpose of the present study was to examine the subcellular distribution of rz51-labeled asialofetuin (AF)’ early after its uptake into isolated rat hepatocytes. Fractionation of isolated rat hepatocytes was carried out using isotonic Nycodenz/sucrose density gradients. The data ob’ Abbreviations used: AF, asialofetuin; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 455
0003-2697184
$3.00
Copyright 0 1984 by Academic press. Inc. All rights of reproduction in any form reserwd
456
KINDBERG
tamed suggest that the ligand passes through two distinct vesicular structures on its way to the lysosomes. MATERIALS
AND
METHODS
Materials. Nycodenz and Maxidens were obtained from Nyegaard and Company (Oslo, Norway). ‘251-Labeled asialofetuin was prepared as described elsewhere ( 16). Collagenase and bovine serum albumin (BSA) were obtained from Sigma Chemical Company (Poole, Dorset, U.K.) All other chemicals were of the purest grades available. Preparation of puriJied hepatocytes. Isolated rat liver cells were prepared from male Wistar rats (200-250 g) by collagenase perfusion (17,18). The total liver cell suspension was incubated for 30 min at 37”C, sieved through nylon mesh, and centrifuged for 30 s at 5Og to pellet the hepatocytes. The pellet was resuspended in ice-cold minimal salt solution containing 1% (w/v) BSA (9) and recentrifuged twice. The resulting cell suspension of almost pure hepatocytes was 90- 100% viable as judged by the trypan blue exclusion test. Binding and internalization of the ligand. The hepatocyte suspension was incubated for 60 min at 4°C in the presence of 1251-labeled asialofetuin (2 &ml) to allow the ligand to bind to cell surface receptors. The ligand is not internalized at this temperature (16). The cells were washed three times in the minimal salt solution with 1% BSA at ice-cold temperature to remove unbound l&and. Cell concentration at this time was 5- 10 X lo6 cells/ml. Aliquots of cell suspension were incubated at 37°C for various time periods to allow internalization of the ligand. Warm incubation medium was added to the icecold aliquot to start the incubation and at the end of the required time period ice-cold medium was added and the mixture kept in ice. After incubation each aliquot was washed in ice-cold 0.25 M sucrose and homogenized in an ice-cold Dounce homogenizer with a tight-fitting pestle (9,lO). The homogenate was centrifuged for 115 s at 2000g in a
ET
AL.
Sorvall SS24 rotor, the supematant was kept, and the pellet was rehomogenized in 0.25 M sucrose. After centrifugation the second supematant was combined with the first. Centrifugation conditions. Preformed gradients were made up in 38-ml centrifuge tubes using a two-chamber mixer. The gradients were formed with 0.25 M sucrose solution containing 1 mM EDTA and 1 mM Hepes (pH 7.2) and a 45% (w/v) Nycodenz solution also containing 1 mM EDTA and 1 mM Hepes (pH 7.2). The resulting gradients had the density range 1.04-l .20 g/ml. The cytoplasmic extract was top loaded onto the gradients (4 ml/gradient) and centrifuged at 4°C at 85,OOOg for the times shown in the text and figures. Each gradient was fmctionated by upward displacement, using Maxidens as the displacement fluid, into 2.0-ml fractions. The density of each fraction was determined using the equation: density = 3.4 In - 3.55, where n is the refractive index at 20°C. This is the equation given for isotonic Nycodenz/sucrose gradients (19) and, in the case of the gradients used here, gives a useful approximation of the density of fractions. ’ The distribution of the ligand was determined by use of a gamma counter, membrane distribution by 5’nucleotidase assay (20), protein by Coomassie blue assay (21), lysosomes by acid phosphatase assay ( lo), and peroxisomes by catalase assay (22). RESULTS
Sedimentation Properties of Newly-Formed Endocytic Vesicles Morphological studies have indicated that asialoglycoproteins during their intracellular transport in hepatocytes are found in vesicles of increasing size (5,8). This change in size is likely to be reflected in variations in their rate of sedimentation in Nycodenz gradients. To determine the centrifugation time needed to get the newly formed (and smallest) endocytic vesicles to their isopycnic distribution in the gradients, cytoplasmic extracts were prepared 1 min after the start of the uptake
SEPARATION
OF ENJXXYTIC
of ‘251-labeled AF at 37°C. The cells had been previously incubated for 60 min with labeled ligand at 4°C as described under Materials and Methods. In preliminary experiments the density distribution of ‘251-labeled AF and various marker enzymes in gradients was determined following application of the cell homogenate on top and at the bottom of the gradient. In the experiment depicted in Fig. 1, the cytoplasmic extracts were prepared after incubating the cells for 1 min at 37°C. The tubes were centrifuged for 1 h at 85,000g. The results showed that the marker enzymes for lysosomes (acid phosphatase), mitochondria (cytochrome ox&se), peroxisomes (catalase), and the plasma membrane (5’nucleotidase) were practically identical for top-loaded and bottom-loaded gradients. Therefore, these organelles had reached their equilibrium density in the gradient. On the other hand, the distributions of rz51-labeled AF did not coincide, indicating that the newly formed endocytic vesicles were not distributed isopycnically. The cells were washed in a buffer
“1.05
1.10 Density
1.15 (g/ml)
FIG. 1. Distribution patterns of *251-labeled AF in Nycodenz gradients after loading postnuclear fractions on top (filled symbols) or at the bottom (open symbols) of the gradients. The postnuclear fraction was prepared from cells that had been preincubated at 4°C for 60 min in the presence of 1251-labeledAF (1 nM) and then, after washing, incubated for I min at 37°C. The tubes were centrifuged for 1 h (circles) or 2 h (squares) at 85,000g. The radioactivities in the gradients are plotted against the density of the fractions. Only radioactivity in the gradient itself is shown.
VESICLES
457
-I
1 2 1.05 Fr. nr.
1.10
1.15
Density
FIG. 2. The effect of centrifugation time on the distribution of 12sI-labeledAF in Nycodenz gradients. Aliquots of postnuclear fractions were prepared from cells that had been given a 4”C-binding period of labeled AF and then incubated at 37°C for 1 min. The postnuclear fractions were layered on top of the gradients and centrifuged at 85,000g for 1 (O), 2 (A), or 3 h (0). Radioactivities were determined in the fractions and are plotted against density of the fractions, except for the two upper fractions which contain material not entering the gradient itself.
containing 4 mM EDTA before homogenization. Hence, only internalized ligand is recovered in the gradients (16). To determine the time needed for the newly formed endocytic vesicles to reach their bouyant density during centrifugation at 85,OOOg, homogenates were again prepared from cells that had been incubated at 37°C for 1 min (after a binding period of ‘251labeled AF at 4°C). The results showed that the distribution of radioactivity migrated further down the gradient with increasing centrifugation times up to 3 h. Figure 2 shows the distribution of ‘251-labeled AF in gradients centrifuged for 1, 2, and 3 h at 85,000g. The cytoplasmic extracts prepared 1 min after the start of the uptake of ‘251-labeled AF were layered on top of the gradients. Increasing the time of centrifugation up to 4 h produced no further migration of the vesicles, indicating that they had reached their isopycnic position after 3 h of centrifugation at 85,000g. Peak radioactivity was then at a density of 1.11 g/ml.
458
KINDBERG
Separation of Endocytic Vesicles Based on Differences in Sedimentation Rate The results in Fig. 2 show that the newly formed endocytic vesicles containing AF slowly move toward their isopycnic position. If the endocytic vesicles subsequently increase in size, such size differences should allow the separation of vesicles at various stages to be carried out. In the experiment depicted in Fig. 3 the cytoplasmic extracts were prepared from cells that were preincubated with ‘251-labeled AF at 4°C and then, after washing, were incubated at 37°C for 30 set, and 1, 2.5, 15, and 30 min. The cytoplasmic extracts were layered on top of the gradients and centrifuged for 45 min at 85,OOOg. The distribution of the ligand (Fig. 3) shows clearly the differences in the sedimentation behavior of the particles after different incubation times. After 30 s the small amount of internalized ligand was all in the form of slowly sedimenting particles. After 1.0 min, the amount of internalized ligand was more than doubled, consisting of a prominent peak of “slow”-sedimenting vesicles and populations of faster-moving particles. After incubation for 2.5 min slow-moving vesicles were still apparent, but much larger populations 0
,
Fr. nr.
1
Density
FIG. 3. Distribution of ‘251-labeled AF containing endocytic vesicles in Nycodenz gradients after short (45 min) centrifugation time. Following incubation with “rIlabeled AF at 4°C for 60 min cells were washed and then incubated at 37°C for 0.5 (O), 1 (A), 2.5 (A), 15 (m), and 30 min (0). Postnuclear fractions were subsequently prepared and layered on top of linear Nycodenz gradients. The tubes were centrifuged for 45 min at 85,OOOg.The results are presented as described in Fig. 2
ET AL.
of the faster-moving vesicles had developed. After 15 min, all the internalized ligand is incorporated in the fast-sedimenting vesicles, as is the reduced amount of ligand present after 30 min of incubation. The results demonstrate that, initially, the vesicles are small and of similar size. With increasing incubation times, more ligand is endocytosed and the vesicles, in subsequent steps, increase in size and, thus, sediment at a faster rate. Recent electron microscopic investigations on the fractions obtained after 45 min of centrifugation in Nycodenz gradients confirm the differences in size between vesicles in fractions with densities of about 1.05 and fractions with densities of about 1.10 g/ml (T. Ford, data to be published). The amount of ligand recovered in the gradients was, as expected, dependent on the time of incubation of the cells. It is known that during incubation of 4°C the ligand is bound to cell surface receptors but not internalized and, when the cells are washed and incubated at 37°C the surface-bound ligand is internalized with a half-time of about 3 min (16). After 15 min all ligand is intemalized. At approximately this time point the AF starts to be degraded (23), and as the degradation products leave the lysosomes as well as the cells very quickly (23,24), the cellassociated radioactivity starts to decrease. Although the amount of cell-associated radioactivity is fairly constant during the first 15 min of incubation, the amount of radioactivity recovered in the gradients increases with incubation time up to 15 min. This is due to the fact that only internalized ligand enters the gradients. Ligand associated with surface receptors is released from the cells when they are washed in calcium-free sucrose solutions before homogenization. Isopycnic Distributions of ‘“I-Labeled AF at Various Times after the Start of the Internalization When cytoplasmic extracts, prepared after various periods of incubation to internalize
SEPARATION
OF ENDOCYTIC
A F:06 7
VESICLES
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FIG. 4 (A) Distribution of ‘2sI-labeled AF after isopycnic centrifugation in Nycodenz gradients of postnuclear fractions. The fractions were prepared from cells that were incubated at 37°C for 1 (A), 2.5 (O), and 15 min (0) following preincubation at 4°C with labeled AF. The centrifugation was carried out for 3 h at 85,000g. Results are presented as described in Fig. 2. (B) Distributions of enzymes after isopycnic centrifugation in a Nycodenz gradient of a postnuclear fraction. The density distributions of catalase (A), 5’-nucleotidase (0), and acid phosphatase (0) are shown. The enzyme activities in the fractions are presented as percentages of total recovered activity in the tube and are plotted against the density of the fraction, excepting the two top fractions which contain material not entering the gradient
the ligand, were centrifuged at 85,000g for 3 h it was found that the density distribution was very similar for vesicles containing newly internalized ligand and those prepared up to 30 min after the start of the internalization of the ligand. Figure 4 depicts the density distributions of ligand that had been internalized into the cells for 1, 2.5, and 15 min. Peak activity is in all cases at about 1.11 g/ ml. Included in the figure are distribution curves for lysosomes (acid phosphatase), plasma membrane (5’-nucleotidase), and peroxisomes (catalase). Do the Endocytic Vesicles Change in Size and/or Density during Centrifugation in Nycodenz Gradients? The possibility that AF-containing vesicles change in size and/or density during centrifugation in Nycodenz gradients was considered. To get information about this, the following experiment was done: Cells were first allowed to internalize ‘251-labeled AF for 1 min at 37°C following the l-h binding period at 4°C. Postnuclear fractions were prepared and centrifuged in Nycodenz gradients for either 45 min or 3 h at 85,000g.
The results were as expected (Fig. 5). After a 45-min centrifugation peak radioactivity was in the gradient at a density of 1.06 g/ml; and after 3 h of centrifugation the peak was at 1.11 g/ml. Fractions 10 and 11 (counted from the top of the gradient) in the gradient centrifuged for 3 h were pooled and diluted with 20 vol of 0.25 M sucrose, and samples of this preparation were then recentrifuged for 45 min in new Nycodenz gradients. The results are depicted in Fig. 6, and show that the vesicles had retained their sedimentation properties fairly well during the centrifugation at 85,000g for 3 h. The peak in the distribution curve is still at about 1.06 g/ml. Efects of Ammonia on the Density Distribution of 12’1-Labeled AF in Nycodenz Gradients Cells were first allowed to bind ligand at 4°C. They were then washed and reincubated at 37°C in the presence or absence of 10 mM NH&l. At various time points after the start of the incubation at 37°C cytoplasmic extracts were prepared and centrifuged for 45 min at 85,000g at 4°C. The results show (Fig. 6) that the presence of NH; does effect
460
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the curves generated by the vesicles after 60 min incubation in the presence or absence of the inhibitor shows that degradation is much slower in the presence of the inhibitor. Our results suggest that the presence of NH: reduces the rate of degradation by acting upon the maturation of the endosomes; the ligand must be located in the fastersedimenting form to be available to the lysosomes. During incubation of the cells at 37°C in the presence or absence of NH:, a reduction in total recovered radioactivity in the gradient could be completely accounted for by a corresponding increase in acid-soluble radioactive degradation products (not shown).
Distribution of Free and Bound Ligand in the Cells
115
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ET AL.
glml
FIG. 5. Effects of centrifugation in Nycodenz gradients on the sedimentation properties of newly formed % labeled AF-containing vesicles. Hepatocytes were incubated for 1 min at 37°C following a l-h binding period at 4’C in the presence of ‘251-labeled AF. Postnuclear fractions were prepared and centrifuged in Nycodenz gradients for 45 min (A) or 3 h (B) at 85,000g. Following fractionation, fractions 10 and 1 I (densities 1.09 and 1.10 g/ml) in the gradient centrifuged for 3 h (B) were pooled and diluted with 20 vol of 0.25 M sucrose. This preparation was recentrifugcd at 85,000g for 45 min (C). The radioactivity in the fractions is plotted against the density of the fractions and is presented as the percentage of total recovered radioactivity in the gradient.
Fig. 7 shows the distribution of free and receptor-bound ligand in the cells 1, 5, and 15 min after the start of the incubation at 37°C. These data, which were obtained by separating free and receptor-bound ligand in a G- 100 column, show that almost all ligand is bound in the “small” vesicle while an increasing fraction of ligand is free in the “large” vesicle. However, even after 15 min,
J
Fr. nr.
the distribution of the ligand. A large proportion of the ligand is retained in the form of slowly sedimenting particles, although the larger, faster-sedimenting species are still present in reduced amounts. Degradation of the ligand still occurs, judging by the reduction of the total 12’1 present with increasing incubation time. However, comparison of
Density
FIG. 6. Effect of NH; on the distribution of lz51lab&xl AF in Nycodenz gradients after a short (45-min) centrifugation. Cells were. first incubated at 4°C for 60 min in the presence of the labeled ligand. They were then washed and incubated at 37°C in the presence (open symbols) or absence (filled symbols) of 10 mM NH:. Postnuclear fractions were prepared after 15 and 30 min and layered on top of Nycodenz gradients, which were subsequently centrifuged for 45 min at 85,000g. The results are presented as described in Fii. 2.
SEPARATION
OF ENDGCYTIC
VESICLES
461
receptor dissociated. A similar intracellular transport of asialoglycoproteins was proposed by Hubbard et al. (5-7). The fractionation method described in this report makes it possible to precisely analyze the kinetic aspects of the intracellular transport of endocytosed ligand. The method should therefore be particularly useful for evaluating the effects of drugs and hormones on the heterophagic process. Studies involving gel filtration showed that most of the ligand was bound in the “early” small vesicle while increasing amounts became free in the larger vesicle. These data are compatible with the finding that pH is Fraction nr. lowered in the endocytic vesicle (15,25) and FIG. 7. Gel filtration in Sephadex G-100 of solubilized that the affinity of receptor for the asialoglycells containing ‘2’I-labeled AF. The cells were first coprotein decreases with decreasing pH (2). incubated at 4°C for 60 min with ‘*“I-labeled AF. They were then washed and reincubated at 37°C for 1, 5, and The results are in agreement with the mor15 min. The cells were chilled and treated with 1% phological data presented by Geuze et al. (8). They found that the ligand was associated Triton X-100 in the presence of an excess of unlabeled AF (to prevent reassociation of ligand to the receptor) with the membrane of the initial small vesicle (4). Bound and free ligand were separted by gel filtration and that it was free in the lumen of the through a Sephadex G-100 column (0.9 X 30 cm): The “CURL” (compartment of uncoupling of column was eluted with a minimal salt solution containing receptor and ligand). Harford et al. (3), using Hepes (pH 7.4). The position of free 12SI-labeled AF after a technique similar to ours, also observed gel filtration (indicated by an arrow) was determined by adding the labeled ligand to solubilized cells and then that the ligand became free in the endocytic running the preparation through the column. We have vesicle. shown previously that the receptor-ligand complex In the presence of ammonia and monensin emerges from the column close to the void volume (26). the ligand seemed to be retained in the “early” vesicle. Both these compounds will about 30% of the ligand is still associated prevent the drop in pH which normally takes with the receptor. place in endocytic vesicles (15,25). It is possible that the acidification of the small enDISCUSSION docytic vesicles is necessary for their develThe results obtained in this study suggest opment (by fusion) into the larger endocytic that lz51-labeled AF internalized into hepa- vesicle. tocytes is transported to the lysosomes ACKNOWLEDGMENTS through at least two types of vesicular strucThe authors are grateful to Ms. Kari Holte for expert tures. Initially the ligand is in a relatively small vesicle, but after 5 min its transfor- technical assistance and to Ms. Ba Gsrbitz for preparing the manuscript. The work was financially supported by mation to larger vesicles is almost complete. the Norwegian Council for Science and the Humanities, The two-step process suggested by the the Nansen Foundation, and the Norwegian Council on present data is in agreement with earlier Cardiovascular Diseases. reports: Geuze et al. (8) showed that asialoREFERENCES glycoproteins were transferred from small vesicles located at the surface of the cell to 1. Ashwell, G., and Harford, J. (1982) Annu. Rev. larger vesicles in which the ligand and the Biochem. 51, 53 l-554.
462
KINDBERG
2. Harford, J., and Ashwell, G. (1982) in The Glycoconjugates (Horowitz, M. I., ed.), Vol. 4, pp. 2755, Academic Press, New York. 3. Harford, J., Wolkoff, A. W., Ashwell, G., and Klausner, R. D. (1983) J. Cell Biol. 96, 1824-1828. 4. Bridges, K., Harford, J., Ashwell, G., and Klausner, R. D. (1982) Proc. Natl. Acad. Sci. (USA) 79, 350-354.
Wall, D. A., Wilson, G., and Hubbard, A. L. (1980) Cell 21, 79-93. 6. Hubbard, A. L., Wilson, G., Ashwell, G., and Stukenbrok, H. (1979) J. Cell Biol. 83, 47-64. 7. Zeitlin, P. L., and Hubbard, A. L. (1982) J. Cell Biol. 92, 634-647. 8. Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F., and Schwartz, A. L. (1983) Cell 5.
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Tolleshaug, H., Berg, T., Frolich, W., and Norum, K. R. (1979) Biochim. Biophys. Acta 585, 7 l-84. IO. Berg, T., Ose, T., Ose, L., and Tolleshaug, H. (1981) Int. J. B&hem. 13, 253-259. 11. Tolleshaug, H., and Berg, T. (198 1) Exp. Cell Res.
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15. Merion, M., and Sly, W. S. (1983) J. Cell Biol. 96, 644-650.
16. Tolleshaug, H. (1981) Znt. J. Biochem. 13, 45-51. 17. Seglen, P. 0. (1976) Methods Cell Biol. 13, 29-83. 18. Berg, T., and Blomhoff, R. (1983) in Iodinated Density Gradient Media (Rickwood, D., ed.), pp. 173-174, IRL Press, Oxford. 19. Rickwood, D. (1983) in Iodinated Density Gradient Media (Rickwood, D., ed.), pp. l-21, IRL Press, Oxford. 20. El-Aaser, A. A., and Reid, E. (1969) Histochem. J. 1,417-437.
21. Bradford, M. M. (1976) Anal. B&hem. 22.
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12. Tolleshaug, H., Biochim. Biophys. Acta, in press. 13. Pittman, R. C., Green, S. R., Attie, A. D., and Steinberg, D. (1979) J. Biol. Chem. 254, 68766879.
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Baudhuin, P., Beaufay, H., Rahman-Li, Y., Sehinger, 0. Z., Wattiaux, R., Jacques, P., and De Duve, C. (1964) Biochem. J. 92, 179-184. Tolleshaug, H., Berg, T., Nilsson, M., and Norum, K. R. (1977) B&him. Biophys. Acta 499,73-84. LaBadie, J. H., Peterson Chapman, K., and Aronson, N. N. (1975) B&hem. J. 152,271-279. Tyco, B., and Maxfield, F. R. (1982) Cell 28, 643651. Blomhoff, R., Tolleshaug, H., and Berg, T. (1982) J. Biol. Chem. 257. 7456-7459.