A thermotolerance screen yields an enriched population of Ydj1-CXXX variants. a) Experimental strategy for probing the entirety of CXXX space that can be modified by yeast GGTase-I. A plasmid library containing all 8,000 Ydj1-CXXX combinations was created and transformed into a yeast strain lacking Ydj1 and FTase (yWS2542, ram1Δ ydj1Δ). The transformed colonies were harvested from multiple plates, pooled, and a representative aliquot used to inoculate cultures that were incubated at permissive and restrictive temperatures (25°C, 37°C, and 42°C, respectively) until saturation. Plasmids isolated from all populations were sequenced using high throughput methods, and data analyzed to determine the relative frequency of each Ydj1-CXXX variant in each population. Graphic created using BioRender.com. b–d) Plots of frequencies of sequences observed in naïve yeast library relative to those observed after cell expansion under b) 25°C, c) 37°C, or d) 42°C conditions. Inset is a magnification of points near the origin.