Uncoating of coated vesicles by yeast hsp70 proteins.
The ability of hsp70 isoenzymes from wild-type and mutant yeast strains to uncoat bovine brain coated vesicles was analyzed and compared with that of the brain uncoating ATPase. Results show that, among the four major cytoplasmic isoenzymes produced in wild-type yeast, almost all of the activity is associated with the SSA1 and SSA2 isoenzymes. The SSB1 and SSB2 isoenzymes have almost no uncoating activity and are not found in the clathrin-hsp70 complexes formed during the uncoating reaction. Using hsp70 mutant yeast strains we find a marked difference in uncoating activity between the SSA1 and SSA2 isoenzymes, although there is only a 3% difference between their amino acid sequences. The SSA4 isoenzyme, which is produced only under stress conditions, has an uncoating activity intermediate between SSA1 and SSA2. These results suggest that the ability of hsp70 isoenzymes to uncoat clathrin-coated vesicles is restricted to certain members of the hsp70 family and can be affected by subtle changes in amino acid sequence. We also investigated the uncoating activity of mixtures of isoenzymes and find that the isoenzyme with lower uncoating activity reduces the activity of the isoenzyme with higher uncoating activity possibly by occupying binding sites on coated vesicles.
Gao
B C
BC
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biosca
J
J
Craig
E A
EA
Greene
L E
LE
Eisenberg
E
E
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*
D006360:Heat-Shock Proteins; D007527:Isoenzymes; D000251:Adenosine Triphosphatases