A role for calcium in stabilizing transport vesicle coats.
Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coat-protomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.
Ahluwalia
J P
JP
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Topp
J D
JD
Weirather
K
K
Zimmerman
M
M
Stamnes
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002474:Cell-Free System; D002614:Chelating Agents / Q000494:pharmacology; D020755:Coat Protein Complex I / Q000737:chemistry; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D020755:Coat Protein Complex I; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D002118:Calcium