Curing of Yeast [URE3] Prion by the Hsp40 Cochaperone Ydj1p Is Mediated by Hsp70

[URE3] is a prion of the yeast Ure2 protein. Hsp40 is a cochaperone that regulates Hsp70 chaperone activity. When overexpressed, the Hsp40 Ydj1p cures yeast of [URE3], but the Hsp40 Sis1p does not. On the basis of biochemical data Ydj1p has been proposed to cure [URE3] by binding soluble Ure2p and preventing it from joining prion aggregates. Here, we mutagenized Ydj1p and find that disrupting substrate binding, dimerization, membrane association, or ability to transfer substrate to Hsp70 had little or no effect on curing. J-domain point mutations that disrupt functional interactions of Ydj1p with Hsp70 abolished curing, and the J domain alone cured [URE3]. Consistent with heterologous J domains possessing similar Hsp70 regulatory activity, the Sis1p J domain also cured [URE3]. We further show that Ydj1p is not essential for [URE3] propagation and that depletion of Ure2p is lethal in cells lacking Ydj1p. Our data imply that curing of [URE3] by overproduced Ydj1p does not involve direct interaction of Ydj1p with Ure2p but rather works through regulation of Hsp70 through a specific J-protein/Hsp70 interaction.