A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes

Abstract

The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins.DOI: http://dx.doi.org/10.7554/eLife.15899.001

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…Of note, the halo associated with a-factor "CASQ", which has a Rce1p / Ste24p CaaX protease cleavage resistant motif that severely compromises a-factor production (~1-2% relative to CVIA), still produced a halo similar to wild type. (32) This is due to the halo assay acting as an extremely sensitive and qualitative method for measuring a-factor production. To better assess the amounts of a-factor produced by mutants, we used a quantitative mating assay.…”

“…(32,50) Through this approach, effects on thermotolerance can be directly related to changes in sequence modification by FTase and not impact on downstream processing steps. A library of Ydj1p mutants with C(x)3X sequences was expressed in yeast, and individual colonies surviving growth at high temperature were identified.…”

“…In this case, post-prenylation processing is not required and is actually deleterious to protein function. (32) The CaaX motif that has served as the de facto consensus motif for prenylation by FTase and GGTase-I was first described over three decades ago, when yeast mating factors, Ras GTPases, and nuclear lamins were found to be lipid modified. (33)(34)(35)(36)(37)(38) While there were limited early investigations into shorter Cxx motifs, (39) investigation of the CaaX sequence has predominately focused on amino acid selectivity within the motif for recognition by FTase and/or GGTase-I.…”

“…(46)(47)(48)(49) In the expansion of our understanding of FTase and GGTase-I substrate recognition, the creation of a novel thermotolerance screen for protein prenylation in yeast has augmented our ability to leverage yeast genetic methodology to identify prenyltransferase substrate sequences. (32) This screen is based on the yeast protein Ydj1p, a cytosolic type I Hsp40 co-chaperone that is required for high-temperature growth. (50)(51)(52)(53) Ydj1p is a shunted CaaX protein, being farnesylated but not proteolyzed and methylated.…”

“…(50)(51)(52)(53) Ydj1p is a shunted CaaX protein, being farnesylated but not proteolyzed and methylated. (32) This crucial feature has allowed direct investigation of protein prenylation without the requirement for concurrent sequence compatibility with Rce1p or Ste24p for subsequent proteolysis.…”