A Role for Calcium in Stabilizing Transport Vesicle Coats

Abstract

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 coatprotomer 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.

Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell

“…We found that treating cells with the membrane-permeable calcium chelator BAPTA-AM led to the dissociation of the COPI vesicle coat protein, coatomer, from the Golgi apparatus (31). Using cell-free Golgi binding assays, we also observed that the addition of BAPTA led to the rapid dissociation of coatomer from previously coated Golgi membranes.…”

Manganese Redistribution by Calcium-stimulated Vesicle Trafficking Bypasses the Need for P-type ATPase Function

“…Accordingly, the addition of CaCl 2 suppresses the vacuole fragmentation phenotype of pmr1⌬ mutants (33). In addition, calcium might also regulate the formation of intraGolgi retrograde transport vesicles as it has been shown to stabilize COPI coat onto the Golgi membrane (67). The addition of CaCl 2 caused a significant increase in the intracellular calcium levels and might account for a permanent induction of retroand anterograde pathways.…”

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

“…In higher eukaryotic cells, some of these processes, or steps thereof, are known to be subject to Ca 2ϩ regulation (1,6). In P. tetraurelia, all steps of exo-and endocytosis are known to be accelerated by increased [Ca 2ϩ ] (72), whereas no information is available on trichocyst biogen- esis.…”