1 TITLE 2 A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent 3 phenotypes 4 Emily R. Hildebrandt, Michael Cheng, Peng Zhao, June H. Kim, Lance Wells, and Walter K. 5 Schmidt* 6 Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, GA 7 30602, USA 8 *corresponding author: Walter K. Schmidt, 706-583-8241 (phone), 706-542-1738 (fax), 9
[email protected] (email) 10 11 COMPETING INTERESTS: none 12 13 Key words: CaaX, isoprenylation, HSP40, chaperone, thermotolerance 14 15 ABBREVIATIONS 16 polyacrylamide gel electrophoresis (PAGE) 17 sodium dodecyl sulfate (SDS) 18 heat shock protein 40 (Hsp40) 19 20 ABSTRACT 21 The modifications occurring to CaaX proteins have largely been established using few reporter 22 molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three 23 coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, 24 and COOH-terminal methylation. Here, we investigated the coupling of these modifications in 25 the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical 26 evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. 27 Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are 28 perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal 29 phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our 30 findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is 31 resistant to post-isoprenylation events. These results expand on the complexity of protein 32 isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of 33 Ydj1p and perhaps other CaaX proteins.