Fig. 4.

Evaluation of CKQX variants as FTase substrates. Enrichment profiles of CKQX sequences in a) Ydj1-based and b) Ras-based screens. c) Thermotolerance and gel-shift assays of Ydj1-CKQX variants provide supporting evidence toward the prenylation of noncanonical CKQX sequences by yeast FTase. Thermotolerance assays were performed by culturing yeast to the same density, applying a 10× serial dilution to the cultures, spotting dilution sets onto YPD-rich media, and incubating at indicated temperatures. Notably, plate-based selection requires a slightly higher temperature to mimic the growth profile observed on liquid-based selection. The strains used were transformants of yWS304 containing the indicated Ydj1-CXXX variant. Gel-shift assays were performed for the indicated Ydj1-CXXX variants in the presence and absence of FTase activity. Total yeast extracts were analyzed by SDS–PAGE and immunoblotting. The strains used were yWS2544 (+FTase) and yWS2542 (ram1Δ; -FTase. Seq, sequence; EF, enrichment factor; %, percent prenylation.