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[Preprint]. 2023 Sep 20:2023.09.19.558494. [Version 1] doi: 10.1101/2023.09.19.558494

Figure 1. Interspecies complementation studies of FTase subunits.

Figure 1.

HsFTase and ScFTase subunits are not interchangeable, but co-expression of both α and β subunits of HsFTase can restore FTase activity in the absence of ScFTase. A) Relationships between yeast and human prenyltransferase subunits. B) The mating assay was used to assess FTase dependent production of a-factor. MATa haploid strains were engineered to express yeast (Sc) and/or human FTase α and β subunits (PGKHs), where subunits were encoded either on plasmids (indicated by brackets) or from chromosomal loci (no brackets). MATa strains were mixed with MATα cells (SM1068), mixes subject to 10-fold dilutions, and dilution mixtures spotted onto minimal media (SD) and SC-lysine (-Lys) media. Growth on SD indicates diploid formation, which is a direct indicator of a-factor mating pheromone production. Growth on -Lys reflects the input of MATa cells and reflects both unmated haploid and mated diploid cells; unmated haploid cells (ram1Δ) grow less well on -Lys due to the absence of FTase activity. Quantitative mating test results are reported below each lane relative to the WT strain (7). Strains used were yWS3276 (1), yWS3277 (2), yWS3278 (3), yWS3408 (4), yWS3280 (5), yWS3282 (6), and yWS3283 (7). C) Gel-shift analysis of Ydj1 using the same strains described in panel B. Total cell lysates were prepared and equivalent protein amounts analyzed by Western blot using anti-Ydj1 antibody. u – unprenylated Ydj1; p – prenylated Ydj1. D) Thermotolerance test of the FTase-deficient strain compared to wildtype and humanized FTase strains described in panel B. Strains used were yWS3276 (1), yWS3282 (6), and yWS3283 (7).