Effect of yeast and human DnaJ homologs on clathrin uncoating by 70 kilodalton heat shock protein.

We recently found that the DnaJ homolog auxilin is required for Hsc70 to uncoat clathrin baskets. In the present study, we investigated the effect of two other DnaJ homologs, YDJ1 from yeast and HDJ1 from humans, on the uncoating activity of Hsc70. Neither YDJ1 nor HDJ1 substituted for auxilin in supporting uncoating. Rather, in the presence of auxilin, both YDJ1 and HDJ1 strongly inhibited uncoating at pH 7 and also prevented the binding of Hsc70 to clathrin baskets at pH 6. Both YDJ1, as shown previously, and HDJ1 catalytically induce polymerization of Hsc70 into large polymers in ATP, and the YDJ1 concentration required to inhibit uncoating was similar to the concentration required for polymerization. However, uncoating was almost completely inhibited even at low concentrations of Hsc70 where only partial polymerization occurs, suggesting that YDJ1 inhibits uncoating not only by polymerizing the Hsc70 but also by some other mechanism as well. The effects of YDJ1 and HDJ1 were completely reversible; when they were removed, the Hsc70 regained full activity. Since both YDJ1 and HDJ1 inhibited the uncoating of clathrin baskets by brain cytosol as well as by purified Hsc70, this could be a physiological phenomenon which could affect other activities of Hsc70 in addition to uncoating.

[1]  M. Cheetham,et al.  Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins. , 1996, The Biochemical journal.

[2]  F. Hartl,et al.  Regulation of the Heat-shock Protein 70 Reaction Cycle by the Mammalian DnaJ Homolog, Hsp40* , 1996, The Journal of Biological Chemistry.

[3]  R. Morimoto,et al.  The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj‐1 have distinct roles in recognition of a non‐native protein and protein refolding. , 1996, The EMBO journal.

[4]  J. Reinstein,et al.  The role of ATP in the functional cycle of the DnaK chaperone system. , 1995, Journal of molecular biology.

[5]  B. Risse,et al.  A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s , 1995, The Journal of cell biology.

[6]  R. Morimoto,et al.  Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ‐1. , 1995, The EMBO journal.

[7]  E. Wilson,et al.  Hormone-dependent Transactivation by the Human Androgen Receptor Is Regulated by a dnaJ Protein (*) , 1995, The Journal of Biological Chemistry.

[8]  E. Craig,et al.  Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44 , 1994, The Journal of cell biology.

[9]  E. Eisenberg,et al.  ATPase activity associated with the uncoating of clathrin baskets by Hsp70. , 1994, The Journal of biological chemistry.

[10]  I. Uno,et al.  YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions , 1994, FEBS letters.

[11]  R. Schekman,et al.  A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome , 1993, The Journal of cell biology.

[12]  R. Schekman,et al.  Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70 , 1993, The Journal of cell biology.

[13]  L. Hendershot,et al.  Interconversion of three differentially modified and assembled forms of BiP. , 1992, The EMBO journal.

[14]  A. Caplan,et al.  Characterization of YDJ1: a yeast homologue of the bacterial dnaJ protein , 1991, The Journal of cell biology.

[15]  P. Silver,et al.  A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein , 1989, The Journal of cell biology.

[16]  C. Georgopoulos,et al.  Initiation of lambda DNA replication with purified host‐ and bacteriophage‐encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins. , 1989, The EMBO journal.