Uncoating of coated vesicles by yeast hsp70 proteins.

B.C Gao,J Biosca,E.A Craig,L.E Greene,E Eisenberg

doi:10.1016/s0021-9258(18)55032-8

Abstract

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.

Highlights

It has recently become clear that thebovine brain uncoating ATPase, which strips clathrin offof bovine brain clathrincoated vesicles is a member of the hsp70 class of heat-shock proteins (Chappell et al, 1986; Ungewickell,1985).This large class of proteins includes both proteins which are present constitutively inthe cell andproteins which are induced during heatshock.All of these proteins bindATP very tightly, andone of their distinguishing properties is their ability to be isolated in an almost pure state with the use of an ATP-agarose affinity column
Using yeast mutants to produce specific hsp7O proteins (Craig and Jacobsen, 1984; Craig and Jacobsen, 1985), we find that these proteins have very different abilities to dissociate clathrin from coated vesicles
Since we were purifying the hsp7O proteins from wild-typeyeast, which contains a number of hsps, this doublet may be due to the presence of a mixture of isoenzymes inour final preparation

Summary

Introduction

It has recently become clear that thebovine brain uncoating ATPase, which strips clathrin offof bovine brain clathrincoated vesicles is a member of the hsp70 class of heat-shock proteins (Chappell et al, 1986; Ungewickell,1985).This large class of proteins includes both proteins which are present constitutively inthe cell andproteins which are induced during heatshock (for reviews, see Craig, 1985and Lindquist, 1986).All of these proteins bindATP very tightly, andone of their distinguishing properties is their ability to be isolated in an almost pure state with the use of an ATP-agarose affinity column. Uncoating Activity of the Yeast hsp70 Protein-We tested the ability of the purified wild-typeyeast hsp7O protein preparation to release clathrin from bovinebrain coated vesicles.

Results

Conclusion