Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes

Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes

^*Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and ^†Department of Morphology, University of Geneva, CH-1211 Geneva, Switzerland

Contributed by Lelio Orci

Abstract

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large (≈300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5′-[γ-thio]triphosphate. Coated vesicles are 40–70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5′-[γ-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.

Footnotes

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 28, 1998.
ABBREVIATIONS:

DOPA,

dioleoylphoshatidic acid;

DOPC,

dioleoylphosphatidylcholine;

DOPE,

dioleoylphosphatidylethanolamine;

DOPS,

dioleoylphosphatidylserine;

NBD-,

7-(nitrobenz 2-oxa-1,3 diazol-4yl)-;

NBD-PE,

1-oleoyl-2-NBD-N-aminododecanoyl-sn-glycero-3-phosphoethanolamine;

PA,

phosphatidic acid;

PIP,

phosphatidylinositol 4-phosphate;

PIP2,

phosphatidylinositol 4,5-bisphosphate;

PLD,

phospholipase D;

GTP-γ-S,

guanosine 5′-[γ-thio]triphosphate;

ER,

endoplasmic reticulum;

PI,

phosphatidylinositol;

POPC/POPE,

1-palmitoyl-2-oleoyl-phosphatidyl choline/1-palmitoyl-2-oleoyl-phosphatidylethanolamine;

PS,

phosphatidylserine;

GDP-β-S,

guanosine 5′-[β-thio] diphosphate