ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes

ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes

Washington University School of Medicine, Department of Internal Medicine, 660 South Euclid Avenue, Box 8125, St. Louis, MO 63110

Contributed by Stuart A. Kornfeld

Abstract

The assembly of clathrin-coated vesicles on Golgi membranes is initiated by the GTP-binding protein ADP ribosylation factor (ARF), which generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex. Once bound, the AP-1 recruits clathrin triskelia, which polymerize to form the coat. We have found that ARF⋅GTP also recruits AP-1 and clathrin onto protein-free liposomes. The efficiency of this process is modulated by the composition of the liposomes, with phosphatidylserine being the most stimulatory phospholipid. There is also a requirement for cytosolic factor(s) other than ARF. Thin-section electron microscopy shows the presence of clathrin-coated buds and vesicles that resemble those formed in vivo. These results indicate that AP-1-containing clathrin-coated vesicles can form in the absence of integral membrane proteins. Thus, ARF⋅GTP, appropriate lipids, and cytosolic factor(s) are the minimal components necessary for AP-1 clathrin-coat assembly.

AP-1 adaptor complex

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: skornfel{at}im.wustl.edu.
ABBREVIATIONS:

ARF,

ADP ribosylation factor;

TGN,

trans-Golgi network;

COP,

coatomer protein;

BFA,

brefeldin A;

CCV,

clathrin-coated vesicle;

PC,

phosphatidylcholine;

PS,

phosphatidylserine;

PA,

phosphatidic acid;

PI,

phosphatidylinositol;

PI4P,

PI 4-phosphate;

PIP2,

PI 4,5-bisphosphate;

PIP3,

PI 3,4,5-triphosphate;

GEF,

guanine nucleotide-exchange factor;

MPRs,

mannose 6-phosphate receptors;

DOPC,

dioleoyl form of pure PC;

DOPE,

dioleoyl form of phosphatidylethanolamine