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Endocytosis without clathrin
Cell Biology
International
Reports,
Vol. 15,
NO.
3
1, 1991
ENDOCYTOSIS WITHOUT CLATHRIN (A MINIREVIEW) Kirsten
Sandvig*
and Bo van Deurs§
*Institute for Cancer Research at the Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway; 'Struttural Cell Biology Unit, Department of Anatomy, The Panum University of Copenhagen, DK-2200 Copenhagen Institute, N, Denmark. A large number of physiological ligands are efficiently endocytosed from clathrin-coated pits at the cell surface, and this pathway has been well characterized (for review, see Brodsky, 1988; Goldstein et al., 1985). Whether also a clathrin-independent endocytic pathway coexists in the same cells has been questioned (Hubbard, 1989; Gruenberg and Howell, 1989). Several lines of evidence (see below) now suggest that uptake of ligands, membrane components and solutes can occur from non-clathrin coated areas at the cell surface. This process, although less efficient, may be of importance for retrieval of membrane as well as for renewal and changes of resident membrane molecules normally not internalized by the more efficient coated pit/coated vesicle pathway. Several morphological studies suggest that ligands are internalized from non-clathrin coated areas of the cell surface. Huet et al. (1980) reported uptake of MHC class I molecules from small uncoated surface invaginations, and similarly, Oliver and Hand (1978) found uptake of horseradish peroxidase in smooth-surfaced vesicles in rat parotid acinar cells. Furthermore, internalization of B-adrenergic receptors in A431 cells (Raposo et al., 1989) and insulin uptake in adipocytes (Smith and Jarett, 1983) may occur from non-coated pits. Also, MHC class II molecules, although not present in coated pits, were slowly internalized (Neefjes et al., 1990). Non-physiological ligands like toxins and viruses may use clathrin-independent endocytosis to gain access to the interior of the cells. Morphological studies suggest that both tetanus and botulinum toxin (Montesano et al. 1982; Tran et al., 1987) as well as Simian Virus 40 (Kartenbeck et al., 1989) are internalized from nonclathrin coated membrane areas. To obtain additional evidence for uptake of ligands by the alternative, clathrin-independent, endocytic pathway, methods to block the clathrin-coated pathway are useful. A number of different treatments affecting uptake 0309-l
651/91/010003-6/$03.00/O
0 1991
Academic
Press Ltd
4
Cell Biology
International
Reports,
Vol. 15, No. 7, 1991
by this pathway have been reported. Larkin et al. (1983) found that hypotonic shock followed by potassium-depletion of cells could remove coated pits from the cell surface. This method was used on Hep-2 cells to show that even in cells with almost no clathrin-coats at the membrane the protein toxin ricin is still endocytosed (Moya et al., 1985) and the two picornaviruses human rhinovirus type 2 and encephalomyocarditis virus are still able to infect the cells (Madshus et al., 1987). Rhinovirus requires endocytosis into an intracellular, acidic compartment to exert its pathogenic effect on cells (Madshus et al., 1984), and the results therefore suggest that ligands internalized by the alternative endocytic pathway end up in acidic endosomes. This is in agreement with the finding of Tran et a1.(1987) who reported that cholera toxin, which seems to be endocytosed from non-coated areas of the membrane, becomes colocalized with az-macroglobulin endocytosed from clathrin-coated pits. So far this is the only report suggesting that ligands taken up by the two pathways may be routed to the same compartment. The possibility still exists that the fate of internalized molecules may depend on the uptake mechanism. Daukas and Zigmond (1985) reported that high osmolarity in the medium surrounding leukocytes blocked receptor-mediated endocytosis but not fluid-phase endocytosis. Similar results were obtained by Oka et al. (1989) who found that in rat hepatocytes hypertonicity inhibited endocytosis of asialoorosomucoid, whereas uptake of lucifer yellow still occurred. However, the response to such treatment may be dependent on the cell type used (Carpentier et al., 1989). Another way to inhibit formation of coated vesicles from coated pits is to acidify the cytosol (Sandvig et al., 1987). At least in some cell types endocytosis does still seem to occur when the clathrin-coated pathway is blocked in this way. Both ricin and the fluid-phase markers lucifer yellow (Sandvig et al. 1987) and horse1989) are internalized radish peroxidase (West et al., under conditions where transferrin-endocytosis is strongly inhibited. In contrast, Davoust et al. (1987) found that low cytosolic pH gave a complete block of fluidThis apparent discrepancy phase endocytosis in BHK cells. could be due to different experimental setups, to the ability to different cell lines used, and perhaps their tolerate such an unphysiological condition as low cytosolit pH. It has been argued that the above-mentioned nonphysiological methods could induce a clathrin-independent pathway that is not funtioning under more normal condi-
Cell Biology
International
Reports,
Vol. 15, No. 1, 199 1
5
also from other types of experitions. However, results ments support the concept that non-clathrin mediated endocytosis is a physiological process. Investigations of the stimulated endocytosis occurring in A431 cells upon addition of epidermal growth factor strongly suggest that the stimulated uptake occurs from non-clathrin coated areas of the membrane. The uptake of ligands (epidermal growth factor and transferrin) internalized from coated and the stimulated uptake of pits is not stimulated, fluid phase marker can, in contrast to the basal uptake, be inhibited by the drug amiloride, suggesting that a different mechanism is operating (West et al., 1989). Furthermore, in Vero cells disruption of actin filaments with cytochalasin D seems to inhibit uptake from nonclathrin coated membrane without affecting the clathrin coated pathway (Sandvig and van Deurs, 1990). Payne et al. (1988) were able to show that clathrin heavy chaindeficient yeast mutants still endocytose a-factor. In fact, yeast lacking both the clathrin heavy and light chain is viable (Payne, 1990; Silveira et al., 1990). An important question is of course: How can vesicles form without clathrin, and what type of structures are involved in the possible uptake from non-clathrin coated membrane? We can only partly answer this question. Energy seems to be required for endocytosis in general (Steinman et al., 1974), but the detailed mechanism behind the vesicle formation is unknown. It is possible that some kind of protein coat is involved although it is not visualized by ordinary electron microscopy. Special surface structures on the cytoplasmic phase of the membrane have been observed in some cell types (Prescott and Brightman, 1976; Somlyo et al., 1971; Peters et al., 1985). However, it is not known whether these structures are involved in endocytosis at all. Recently we used Au-labeled Concananvalin A to label the early endocytic vesicles formed in Hep 2 cells, A431 cells and T47D cells, and the data show that the preendosomal compartment comprises both clathrin-coated and noncoated endocytic vesicles with about the same frequenof the non-coated vesicles is slightly cy* The diameter smaller (95nm) than the average clathrin-coated vesicle (110 nm) (Hansen et al., submitted). However, it is still not clear whether such a population of vesicles will be found in all cell types and whether the type of vesicle formed could depend on the culture conditions. It is possible that vesicles formed in growth factor stimulated cells are of a different type than the small vesicles here observed. Only future reseach can clarify these questions.
6
Cell Biology
International
Reports,
Vol. 75, No. 7, 7997
ACKNOWLEDGEMENTS Work from our laboratories referred to in this paper has been supported by grants from The Norwegian and Danish Cancer Societies, The Danish Medical Research Counsil, The NOVO Foundation, and by a NATO Collaborative Research Grant (CRG 900517). REFERENCES Brodsky, F.M.(1988) Living with clathrin: Its role in intracellular membrane traffic. Science 242, 13861402. Carpentier, J.-L., F. Sawano, D. Geiger, P. Gorden, A. Perrelet and Orci, L. (1989) Potassium depletion and hypertonic medium reduce t'non-coated" and clathrincoated pit formation, as well as endocytosis through these two gates. J. Cell. Physiol. 138, 519-526. Daukas, G. and Zigmond, S.H. (1985) Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes. J. Cell Biol. 101, 1673-1679. Davoust, J., J. Gruenberg and Howell, K.E. (1989) Two threshold values of low pH block endocytosis at different stages. EMBO. J. 5, 3601-3609. Goldstein, J.L., M.S. Brown, R.G. Anderson, D.W. Russell endocyand Schneider, W.J. (1985) Receptor-mediated tosis: concepts emerging from receptor system. Annu. Rev. Cell Biol. 1, l-39. Gruenberg, J. and Howell, K.E. (1989) Membrane traffic in endocytosis: Insights from cell-free assays Annu. 5, 453-481. Rev. Cell Biol. Hansen, S.H., Sandvig, K. and van Deurs, B. The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations. Submitted. Hubbard, A.L. (1989) Endocytosis. Current opinions in cell biology 1, 675-683. and Helenius, A. (1989) Kartenbeck, J., H. Stukenbrok Endocytosis of Simian Virus 40 into the endoplasmic J. Cell Biol. 109, 2721-2729. reticulum. and Anderson, Larkin, J.M., M.S. Brown, J.L. Goldstein R.G.W. (1983) Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell 33, 273-285. S. Olsnes and Sandvig, K. 1984. Different Madshus, I.H., pH requirements for entry of the two picornaviruses, human rhinovirus 2 and murine encephalomyocarditis virus. Virology 139, 346-357. Madshus, I.H., K. Sandvig, S. Olsnes and van Deurs, B. (1987) Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses. J. Cell Physiol. 131, 14-22. L. (1982) Montesano, R., J. Roth, A. Robert and Orci,
Cell Biology
International
Reports,
Vol. 75, No. 1, 7991
7
Non-coated membrane invaginations are involved in binding internalization of cholera and tetanus toxins. Nature 296:651-653. Moya, M., A. Dautry-Varsat, B. Goud, D. Louvard and Inhibition of coated pit formaBoguet, P. (1985). tion in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin. J. Cell Biol. 101, 548-559s Neefjes, J.J., V. Stollorz, P.J. Peters, H.J. Geuze and pathway of MHC Ploegh, H.L. (1990) The biosynthetic class II but not class I molecules intersects the endocytic route. Cell 6l, 171-183. Oka, J.A., M.D. Christensen and Weigel, P.H. (1989) Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes. J. Biol. Chem. 264, 12016-12024. Oliver, C. and Hand, A.R. (1978) Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells. J. Cell Biol. 76, 207-220. Payne, G.S. (1990) Genetic Analysis of clathrin function in yeast. J. Membr. Biol. 116, 93-105. Payne, G-S., D. Baker, E. van Tuinen and Schekman, R. (1988) Protein transport to the vacuole and receptormediated endocytosis by clathrin heavy chaindeficient yeast. J. Cell Biol. 106, 1453-1461. Peters, K.R., W.W. Carley and Palade, G.E. (1985) Endothelial plasmalemmal vesicles have a characteristic bipolar surface structure. J. Cell Biol. 101, 22332238. Prescott, L. and Brightman, M.W. (1976) The sarcolemma of aplysia smooth muscle in freeze-fracture preparations. Tissue and Cell 8:241-258. Raposo, G., I. Dunia, C.D. Delavier-Klutchko, S. Kaveri, A.D. Strosberg and Benedetti, E.L. (1989) Internalization of P-adrenergic receptors in A431 cells involves non-coated vesicles. Eur. J. Cell Biol. 50, 340-352. Sandvig, K., S. Olsnes, O.W. Petersen and van Deurs, B. (1987). Acidification of the cytosol inhibits endocytosis from coated pits. J. Cell Biol. 105, 679689. Sandvig, K. and van Deurs, B. (1990) Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J. Biol. Chem. 265~6382-6388. Silveira, L.A., D.H. Wong, F.R. Masiarz and Schekman, R. 1990. Yeast clathrin has a distinct light chain that is important for cell growth. J. Cell Biol. 111, 1437-1449. Smith, R.M. and Jarett, L. (1983) Quantitative ultrastructural analysis of receptor-mediated insulin uptake into adipocytes. J. Cell Physiol. 115, 199
8
Cell Biology
International
Reports,
Vol. 15, No. 1, 1991
-207.
Somlyo, A.P., C.E. Devine, A.V. Somlyo and North, S.R. (1971) Sarcoplasmic reticulum and the temperaturedependent contraction of smooth muscle in calciumfree solutions. J. Cell Biol. 5l, 722-741. Steinman, R.M., J.M. Silver and Cohn, Z.A. (1974). Pinocytosis in fibroblasts. Quantitative studies in vitro. J. Cell Biol. 63, 949-969. Tran, D., J.L. Carpentier, F. Sawano, P. Gorden and Orci, L. (1987) Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc. Natl. Acad. Sci. U. S. A. 84, 7957-7961. West, M.A., M.S. Bretscher and Watts, C. (1989) Distinct endocytic pathways in epidermal growth factorstimulated human carcinoma A431 cells. J. Cell Biol. 109, 2731-2739. Paper
received
Og.ll.gO.
Paper
accepted
16.11.90.
International
Reports,
Vol. 15,
NO.
3
1, 1991
ENDOCYTOSIS WITHOUT CLATHRIN (A MINIREVIEW) Kirsten
Sandvig*
and Bo van Deurs§
*Institute for Cancer Research at the Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway; 'Struttural Cell Biology Unit, Department of Anatomy, The Panum University of Copenhagen, DK-2200 Copenhagen Institute, N, Denmark. A large number of physiological ligands are efficiently endocytosed from clathrin-coated pits at the cell surface, and this pathway has been well characterized (for review, see Brodsky, 1988; Goldstein et al., 1985). Whether also a clathrin-independent endocytic pathway coexists in the same cells has been questioned (Hubbard, 1989; Gruenberg and Howell, 1989). Several lines of evidence (see below) now suggest that uptake of ligands, membrane components and solutes can occur from non-clathrin coated areas at the cell surface. This process, although less efficient, may be of importance for retrieval of membrane as well as for renewal and changes of resident membrane molecules normally not internalized by the more efficient coated pit/coated vesicle pathway. Several morphological studies suggest that ligands are internalized from non-clathrin coated areas of the cell surface. Huet et al. (1980) reported uptake of MHC class I molecules from small uncoated surface invaginations, and similarly, Oliver and Hand (1978) found uptake of horseradish peroxidase in smooth-surfaced vesicles in rat parotid acinar cells. Furthermore, internalization of B-adrenergic receptors in A431 cells (Raposo et al., 1989) and insulin uptake in adipocytes (Smith and Jarett, 1983) may occur from non-coated pits. Also, MHC class II molecules, although not present in coated pits, were slowly internalized (Neefjes et al., 1990). Non-physiological ligands like toxins and viruses may use clathrin-independent endocytosis to gain access to the interior of the cells. Morphological studies suggest that both tetanus and botulinum toxin (Montesano et al. 1982; Tran et al., 1987) as well as Simian Virus 40 (Kartenbeck et al., 1989) are internalized from nonclathrin coated membrane areas. To obtain additional evidence for uptake of ligands by the alternative, clathrin-independent, endocytic pathway, methods to block the clathrin-coated pathway are useful. A number of different treatments affecting uptake 0309-l
651/91/010003-6/$03.00/O
0 1991
Academic
Press Ltd
4
Cell Biology
International
Reports,
Vol. 15, No. 7, 1991
by this pathway have been reported. Larkin et al. (1983) found that hypotonic shock followed by potassium-depletion of cells could remove coated pits from the cell surface. This method was used on Hep-2 cells to show that even in cells with almost no clathrin-coats at the membrane the protein toxin ricin is still endocytosed (Moya et al., 1985) and the two picornaviruses human rhinovirus type 2 and encephalomyocarditis virus are still able to infect the cells (Madshus et al., 1987). Rhinovirus requires endocytosis into an intracellular, acidic compartment to exert its pathogenic effect on cells (Madshus et al., 1984), and the results therefore suggest that ligands internalized by the alternative endocytic pathway end up in acidic endosomes. This is in agreement with the finding of Tran et a1.(1987) who reported that cholera toxin, which seems to be endocytosed from non-coated areas of the membrane, becomes colocalized with az-macroglobulin endocytosed from clathrin-coated pits. So far this is the only report suggesting that ligands taken up by the two pathways may be routed to the same compartment. The possibility still exists that the fate of internalized molecules may depend on the uptake mechanism. Daukas and Zigmond (1985) reported that high osmolarity in the medium surrounding leukocytes blocked receptor-mediated endocytosis but not fluid-phase endocytosis. Similar results were obtained by Oka et al. (1989) who found that in rat hepatocytes hypertonicity inhibited endocytosis of asialoorosomucoid, whereas uptake of lucifer yellow still occurred. However, the response to such treatment may be dependent on the cell type used (Carpentier et al., 1989). Another way to inhibit formation of coated vesicles from coated pits is to acidify the cytosol (Sandvig et al., 1987). At least in some cell types endocytosis does still seem to occur when the clathrin-coated pathway is blocked in this way. Both ricin and the fluid-phase markers lucifer yellow (Sandvig et al. 1987) and horse1989) are internalized radish peroxidase (West et al., under conditions where transferrin-endocytosis is strongly inhibited. In contrast, Davoust et al. (1987) found that low cytosolic pH gave a complete block of fluidThis apparent discrepancy phase endocytosis in BHK cells. could be due to different experimental setups, to the ability to different cell lines used, and perhaps their tolerate such an unphysiological condition as low cytosolit pH. It has been argued that the above-mentioned nonphysiological methods could induce a clathrin-independent pathway that is not funtioning under more normal condi-
Cell Biology
International
Reports,
Vol. 15, No. 1, 199 1
5
also from other types of experitions. However, results ments support the concept that non-clathrin mediated endocytosis is a physiological process. Investigations of the stimulated endocytosis occurring in A431 cells upon addition of epidermal growth factor strongly suggest that the stimulated uptake occurs from non-clathrin coated areas of the membrane. The uptake of ligands (epidermal growth factor and transferrin) internalized from coated and the stimulated uptake of pits is not stimulated, fluid phase marker can, in contrast to the basal uptake, be inhibited by the drug amiloride, suggesting that a different mechanism is operating (West et al., 1989). Furthermore, in Vero cells disruption of actin filaments with cytochalasin D seems to inhibit uptake from nonclathrin coated membrane without affecting the clathrin coated pathway (Sandvig and van Deurs, 1990). Payne et al. (1988) were able to show that clathrin heavy chaindeficient yeast mutants still endocytose a-factor. In fact, yeast lacking both the clathrin heavy and light chain is viable (Payne, 1990; Silveira et al., 1990). An important question is of course: How can vesicles form without clathrin, and what type of structures are involved in the possible uptake from non-clathrin coated membrane? We can only partly answer this question. Energy seems to be required for endocytosis in general (Steinman et al., 1974), but the detailed mechanism behind the vesicle formation is unknown. It is possible that some kind of protein coat is involved although it is not visualized by ordinary electron microscopy. Special surface structures on the cytoplasmic phase of the membrane have been observed in some cell types (Prescott and Brightman, 1976; Somlyo et al., 1971; Peters et al., 1985). However, it is not known whether these structures are involved in endocytosis at all. Recently we used Au-labeled Concananvalin A to label the early endocytic vesicles formed in Hep 2 cells, A431 cells and T47D cells, and the data show that the preendosomal compartment comprises both clathrin-coated and noncoated endocytic vesicles with about the same frequenof the non-coated vesicles is slightly cy* The diameter smaller (95nm) than the average clathrin-coated vesicle (110 nm) (Hansen et al., submitted). However, it is still not clear whether such a population of vesicles will be found in all cell types and whether the type of vesicle formed could depend on the culture conditions. It is possible that vesicles formed in growth factor stimulated cells are of a different type than the small vesicles here observed. Only future reseach can clarify these questions.
6
Cell Biology
International
Reports,
Vol. 75, No. 7, 7997
ACKNOWLEDGEMENTS Work from our laboratories referred to in this paper has been supported by grants from The Norwegian and Danish Cancer Societies, The Danish Medical Research Counsil, The NOVO Foundation, and by a NATO Collaborative Research Grant (CRG 900517). REFERENCES Brodsky, F.M.(1988) Living with clathrin: Its role in intracellular membrane traffic. Science 242, 13861402. Carpentier, J.-L., F. Sawano, D. Geiger, P. Gorden, A. Perrelet and Orci, L. (1989) Potassium depletion and hypertonic medium reduce t'non-coated" and clathrincoated pit formation, as well as endocytosis through these two gates. J. Cell. Physiol. 138, 519-526. Daukas, G. and Zigmond, S.H. (1985) Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes. J. Cell Biol. 101, 1673-1679. Davoust, J., J. Gruenberg and Howell, K.E. (1989) Two threshold values of low pH block endocytosis at different stages. EMBO. J. 5, 3601-3609. Goldstein, J.L., M.S. Brown, R.G. Anderson, D.W. Russell endocyand Schneider, W.J. (1985) Receptor-mediated tosis: concepts emerging from receptor system. Annu. Rev. Cell Biol. 1, l-39. Gruenberg, J. and Howell, K.E. (1989) Membrane traffic in endocytosis: Insights from cell-free assays Annu. 5, 453-481. Rev. Cell Biol. Hansen, S.H., Sandvig, K. and van Deurs, B. The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations. Submitted. Hubbard, A.L. (1989) Endocytosis. Current opinions in cell biology 1, 675-683. and Helenius, A. (1989) Kartenbeck, J., H. Stukenbrok Endocytosis of Simian Virus 40 into the endoplasmic J. Cell Biol. 109, 2721-2729. reticulum. and Anderson, Larkin, J.M., M.S. Brown, J.L. Goldstein R.G.W. (1983) Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell 33, 273-285. S. Olsnes and Sandvig, K. 1984. Different Madshus, I.H., pH requirements for entry of the two picornaviruses, human rhinovirus 2 and murine encephalomyocarditis virus. Virology 139, 346-357. Madshus, I.H., K. Sandvig, S. Olsnes and van Deurs, B. (1987) Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses. J. Cell Physiol. 131, 14-22. L. (1982) Montesano, R., J. Roth, A. Robert and Orci,
Cell Biology
International
Reports,
Vol. 75, No. 1, 7991
7
Non-coated membrane invaginations are involved in binding internalization of cholera and tetanus toxins. Nature 296:651-653. Moya, M., A. Dautry-Varsat, B. Goud, D. Louvard and Inhibition of coated pit formaBoguet, P. (1985). tion in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin. J. Cell Biol. 101, 548-559s Neefjes, J.J., V. Stollorz, P.J. Peters, H.J. Geuze and pathway of MHC Ploegh, H.L. (1990) The biosynthetic class II but not class I molecules intersects the endocytic route. Cell 6l, 171-183. Oka, J.A., M.D. Christensen and Weigel, P.H. (1989) Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes. J. Biol. Chem. 264, 12016-12024. Oliver, C. and Hand, A.R. (1978) Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells. J. Cell Biol. 76, 207-220. Payne, G.S. (1990) Genetic Analysis of clathrin function in yeast. J. Membr. Biol. 116, 93-105. Payne, G-S., D. Baker, E. van Tuinen and Schekman, R. (1988) Protein transport to the vacuole and receptormediated endocytosis by clathrin heavy chaindeficient yeast. J. Cell Biol. 106, 1453-1461. Peters, K.R., W.W. Carley and Palade, G.E. (1985) Endothelial plasmalemmal vesicles have a characteristic bipolar surface structure. J. Cell Biol. 101, 22332238. Prescott, L. and Brightman, M.W. (1976) The sarcolemma of aplysia smooth muscle in freeze-fracture preparations. Tissue and Cell 8:241-258. Raposo, G., I. Dunia, C.D. Delavier-Klutchko, S. Kaveri, A.D. Strosberg and Benedetti, E.L. (1989) Internalization of P-adrenergic receptors in A431 cells involves non-coated vesicles. Eur. J. Cell Biol. 50, 340-352. Sandvig, K., S. Olsnes, O.W. Petersen and van Deurs, B. (1987). Acidification of the cytosol inhibits endocytosis from coated pits. J. Cell Biol. 105, 679689. Sandvig, K. and van Deurs, B. (1990) Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J. Biol. Chem. 265~6382-6388. Silveira, L.A., D.H. Wong, F.R. Masiarz and Schekman, R. 1990. Yeast clathrin has a distinct light chain that is important for cell growth. J. Cell Biol. 111, 1437-1449. Smith, R.M. and Jarett, L. (1983) Quantitative ultrastructural analysis of receptor-mediated insulin uptake into adipocytes. J. Cell Physiol. 115, 199
8
Cell Biology
International
Reports,
Vol. 15, No. 1, 1991
-207.
Somlyo, A.P., C.E. Devine, A.V. Somlyo and North, S.R. (1971) Sarcoplasmic reticulum and the temperaturedependent contraction of smooth muscle in calciumfree solutions. J. Cell Biol. 5l, 722-741. Steinman, R.M., J.M. Silver and Cohn, Z.A. (1974). Pinocytosis in fibroblasts. Quantitative studies in vitro. J. Cell Biol. 63, 949-969. Tran, D., J.L. Carpentier, F. Sawano, P. Gorden and Orci, L. (1987) Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc. Natl. Acad. Sci. U. S. A. 84, 7957-7961. West, M.A., M.S. Bretscher and Watts, C. (1989) Distinct endocytic pathways in epidermal growth factorstimulated human carcinoma A431 cells. J. Cell Biol. 109, 2731-2739. Paper
received
Og.ll.gO.
Paper
accepted
16.11.90.