Interaction of clathrin with large unilamellar phospholipid vesicles at neutral pH. Lipid dependence and protein penetration.
The interaction of clathrin with large unilamellar vesicles of various lipid compositions has been examined at neutral pH. Clathrin induces leakage of contents of vesicles that contain the acidic phospholipid phosphatidylserine. Leakage is greatly enhanced by the presence of a relatively minor amount of cholesterol, but is inhibited by phosphatidylcholine. Resonance energy transfer measurements between tryptophan residues of the protein and a fluorescent lipid analog, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine incorporated into the liposomal bilayer, suggests a dynamic interaction of clathrin with the bilayer at neutral pH. This interaction includes a (partial) penetration of the protein into the lipid bilayer, as revealed by hydrophobic photoaffinity labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine. The interaction of clathrin with lipid vesicles at neutral pH is inhibited when the protein is pretreated with trypsin or with the reducing agent dithiothreitol, suggesting that structural requirements govern clathrin-membrane interaction at these conditions. The physiological relevance of the present observations in light of vesiculation and endosomal maturation is discussed.
Seppen
J
J
Laboratory of Physiological Chemistry, University of Groningen, Netherlands.
Ramalho-Santos
J
J
de Carvalho
A P
AP
ter Beest
M
M
Kok
J W
JW
de Lima
M C
MC
Hoekstra
D
D
D000345:Affinity Labels; D001345:Autoradiography; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010777:Photochemistry; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D002966:Clathrin; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D010743:Phospholipids; D014357:Trypsin; D004229:Dithiothreitol