Effect of Yeast and Human DnaJ Homologs on Clathrin Uncoating by 70 Kilodalton Heat Shock Protein

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Abstract
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.