Acute cholesterol depletion inhibits clathrin-coated pit budding

A Subtil

I Gaidarov

K Kobylarz

M Lampson

J Keen

T McGraw

^{Department of Biochemistry, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021; and}^{Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107}

^{A.S. and I.G. contributed equally to this work.}

^{To whom reprint requests should be addressed. e-mail:}ude.llenroc.dem.liam@wargcmet.

Communicated by David D. Sabatini, New York University School of Medicine, New York, NY

Received 1999 Feb 25; Accepted 1999 Apr 15.

Abstract

Many biologically important macromolecules are internalized into cells by clathrin-coated pit endocytosis. The mechanism of clathrin-coated pit budding has been investigated intensively, and considerable progress has been made in characterizing the proteins involved in internalization. Membrane lipid composition and the lateral organization of lipids and proteins within membranes are believed to play an important role in the regulation of membrane-trafficking processes. Here we report that membrane cholesterol plays a critical role in clathrin-coated pit internalization. We show that acute cholesterol depletion, using β-methyl-cyclodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green fluorescent protein–clathrin conjugate in living cells. Ultrastructural studies indicate that acute cholesterol depletion causes accumulation of flat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possibility that flat clathrin lattices are direct precursors of indented pits and endocytic vesicles in intact cells. We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.

Keywords: endocytosis, internalization

Abstract

The uptake of membrane through clathrin-coated pits is one of the most important and intensively studied internalization mechanisms in cells (reviewed in ref. 1). Clathrin-mediated internalization involves the assembly of clathrin and its associated adaptin (AP-2) complex on the plasma membrane, concentration of membrane proteins in clathrin-coated pits, budding of the coated pits from the membrane, release of clathrin and AP-2 from the vesicles, and reformation of new coated pits. In addition to clathrin and the AP-2 complex, a number of proteins involved in clathrin-coated pit formation and budding have been identified, and progress has been made in characterizing their roles in internalization (2–4).

A number of studies have focused on the role of lipids in regulating membrane trafficking (reviewed in ref. 5). In regard to clathrin-mediated internalization, AP-2 binds phosphatidylinositides, and this binding may play a role in recruiting AP-2 to membranes as well as in affecting its function (e.g., refs. 6 and 7). Dynamin, a GTPase that is involved in the budding of clathrin-coated vesicles, binds acidic phospholipids, and this binding may play a role in recruiting or regulating the activity of dynamin (8, 9). Thus, the local lipid environment may be important in regulating the assembly and function of clathrin-coated pits.

There is evidence that a lateral organization of lipids in cholesterol- and sphingolipid-rich domains is involved in trafficking processes (10–15). Most direct evidence for the function of these domains in membrane traffic comes from studies of glycosylphosphatidylinositol (GPI)-modified proteins. For example, the concentration of GPI-linked proteins in these cholesterol- and sphingolipid-rich domains is believed to be a mechanism for targeting GPI-linked proteins to the apical membrane from the trans-Golgi network (e.g., ref. 16). One frequently used approach to examine the function of cholesterol has been to deplete or sequester cholesterol (14, 16–20). To examine the role of cholesterol in clathrin-mediated internalization, we studied the effect of acute cholesterol depletion, using β-methyl-cyclodextrin (CD), on the endocytic behavior of the transferrin receptor (TR). The trafficking of the TR has been well characterized in a number of different cell types, and it is used often as a marker of clathrin-mediated endocytosis. Here we report that acute cholesterol depletion results in a marked reduction in the rate of TR internalization. This effect is a result of a corresponding decrease in detachment of endocytic-coated pits, visualized by using a green fluorescent protein (GFP)–clathrin conjugate in living cells. Electron microscopy analysis indicates that cholesterol depletion acts by preventing coated pit budding.

Acknowledgments

We thank William Mallet, Fred Maxfield, and Tim Ryan for their thoughtful comments. We thank Neelima Shah of the Diabetes and Endocrinology Research Center and the Biomedical Imaging Core Laboratory of the University of Pennsylvania for electron microscopy services. This work was supported by National Institutes of Health Grants DK52852 (T.E.M.) and GM28526 (J.H.K.) and by a fellowship program sponsored by the Charles H. Revson Foundation (A.S.).

Acknowledgments

ABBREVIATIONS

TR	transferrin receptor
CD	β-methyl-cyclodextrin
GPI	glycosylphosphatidylinositol
GFP	green fluorescent protein, CHO, Chinese hamster ovary
AP	adaptin

ABBREVIATIONS

Note Added in Proof

While this manuscript was in review, another paper presenting similar results was published (47).

Note Added in Proof

References

1. Mukherjee S, Ghosh R N, Maxfield F R. Physiol Rev. 1997;77:759–803.[PubMed]
2. Schmid S L. Trends Cell Biol. 1993;3:145–148.[PubMed]
3. Moore M S, Mahaffey D T, Brodsky F M, Anderson R G. Science. 1987;236:558–563.[PubMed]
4. Takei K, Haucke V, Slepnev V, Farsad K, Salazar M, Chen H, De Camilli P. Cell. 1998;94:131–141.[PubMed]
5. De Camilli P, Emr S D, McPherson P S, Novick P. Science. 1996;271:1533–1539.[PubMed]
6. Gaidarov I, Chen Q, Falck J R, Reddy K K, Keen J H. J Biol Chem. 1996;271:20922–20929.[PubMed]
7. Rapoport I, Miyazaki M, Boll W, Duckworth B, Cantley L C, Shoelson S, Kirchhausen T. EMBO J. 1997;16:2240–2250.
8. Tuma P L, Stachniak M C, Collins C A. J Biol Chem. 1993;268:17240–17246.[PubMed]
9. Liu J P, Powell K A, Sudhof T C, Robinson P J. J Biol Chem. 1994;269:21043–21050.[PubMed]
10. Rothblat G H, Mahlberg F H, Johnson W J, Phillips M C. J Lipid Res. 1992;33:1091–1097.[PubMed]
11. Schroeder F, Jefferson J R, Kier A B, Knittel J, Scallen T J, Wood W G, Hapala I. Proc Soc Exp Biol Med. 1991;196:235–252.[PubMed]
12. Schroeder F, Woodford J K, Kavecansky J, Wood W G, Joiner C. Mol Membr Biol. 1995;12:113–119.[PubMed]
13. Simons K, van Meer G. Biochemistry. 1988;27:6197–6202.[PubMed]
14. Anderson R G, Kamen B A, Rothberg K G, Lacey S W. Science. 1992;255:410–411.[PubMed]
15. Simons K, Ikonen E. Nature (London) 1997;387:569–572.[PubMed]
16. Scheiffele P, Roth M G, Simons K. EMBO J. 1997;16:5501–5508.
17. Hannan L A, Edidin M. J Cell Biol. 1996;133:1265–1276.
18. Mayor S, Sabharanjak S, Maxfield F R. EMBO J. 1998;17:4626–4638.
19. Kilsdonk E P, Yancey P G, Stoudt G W, Bangerter F W, Johnson W J, Phillips M C, Rothblat G H. J Biol Chem. 1995;270:17250–17256.[PubMed]
20. Neufeld E B, Cooney A M, Pitha J, Dawidowicz E A, Dwyer N K, Pentchev P G, Blanchette-Mackie E J. J Biol Chem. 1996;271:21604–21613.[PubMed]
21. McGraw T E, Greenfield L, Maxfield F R. J Cell Biol. 1987;105:207–214.
22. Johnson L S, Dunn K W, Pytowski B, McGraw T E. Mol Biol Cell. 1993;4:1251–1266.
23. Johnson A O, Ghosh R N, Dunn K W, Garippa R, Park J, Mayor S, Maxfield F R, McGraw T E. J Cell Biol. 1996;135:1749–1762.
24. Mukherjee S, Zha X, Tabas I, Maxfield F R. Biophys J. 1998;75:1915–1925.
25. Gaidarov I, Santini F, Warren R A, Keen J H. Nat Cell Biol. 1999;1:1–7.[PubMed]
26. Santini F, Marks M S, Keen J H. Mol Biol Cell. 1998;9:1177–1194.
27. Yamashiro D J, Tycko B, Fluss S R, Maxfield F R. Cell. 1984;37:789–800.[PubMed]
28. Dautry-Varsat A, Ciechanover A, Lodish H F. Proc Natl Acad Sci USA. 1983;80:2258–2262.
29. Heuser J. J Cell Biol. 1980;84:560–583.
30. Maupin P, Pollard T D. J Cell Biol. 1983;96:51–62.
31. Larkin J M, Brown M S, Goldstein J L, Anderson R G. Cell. 1983;33:273–285.[PubMed]
32. Smythe E, Pypaert M, Lucocq J, Warren G. J Cell Biol. 1989;108:843–853.
33. Lin H C, Moore M S, Sanan D A, Anderson R G. J Cell Biol. 1991;114:881–891.
34. Mahaffey D T, Moore M S, Brodsky F M, Anderson R G. J Cell Biol. 1989;108:1615–1624.
35. Santini F, Keen J H. J Cell Biol. 1996;132:1025–1036.
36. Orlandi P A, Fishman P H. J Cell Biol. 1998;141:905–915.
37. Schnitzer J E, Oh P, Pinney E, Allard J. J Cell Biol. 1994;127:1217–1232.
38. Keen J H, Maxfield F R, Hardegree M C, Habig W H. Proc Natl Acad Sci USA. 1982;79:2912–2916.
39. Moya M, Dautry-Varsat A, Goud B, Louvard D, Boquet P. J Cell Biol. 1985;101:548–559.
40. Tran D, Carpentier J L, Sawano F, Gorden P, Orci L. Proc Natl Acad Sci USA. 1987;84:7957–7961.
41. Brown R E. J Cell Sci. 1998;111:1–9.
42. Anderson R G. Proc Natl Acad Sci USA. 1993;90:10909–10913.
43. Turek J J, Leamon C P, Low P S. J Cell Sci. 1993;106:423–430.[PubMed]
44. Smart E J, Mineo C, Anderson R G. J Cell Biol. 1996;134:1169–1177.
45. Keller P, Simons K. J Cell Biol. 1998;140:1357–1367.
46. Dai J, Ting-Beall H P, Sheetz M P. J Gen Physiol. 1997;110:1–10.
47. Rodal S K, Skretting G, Garred O, Vilhardt F, van Deurs B, Sandvig K. Mol Biol Cell. 1999;10:961–974.