Certain essential proteins that control cell cycle progression are regulated by degradation. Rapid degradation of the activator of the cell cycle START checkpoint, Cln3 cyclin, is triggered by its phosphorylation. Evidence has been presented that this phosphorylation event requires binding of the yeast molecular chaperon Ydj1 to Cln3. The goal of the proposed studies is to understand the novel function of the chaperons Ydj1 and Hsp70 in protein phosphorylation through enzymological studies and in vitro reconstitution of Cln3 phosphorylation. It will be analyzed whether 1) Ydj1, by binding to Cln3, exposes the otherwise inaccessible site of phosphorylation, and whether Ydj1 promotes association of Cln3 with the kinase; 2) how Hsp70s cooperate with Ydj1 in Cln3 phosphorylation; 3) whether chaperons function in this regulation as sensors of heat shock and other stresses and whether they are rate-limiting factors of Cln3 phosphorylation; 4) whether chaperons play a general role in phosphorylation of a variety of regulatory proteins upon exposure of cells to stresses which cause the accumulation of damaged polypeptide.
