Figure 8. Prenylation studies can be coupled with CaaX proteolysis studies to better understand CaaX PTMs.

The mating assay was performed as described in Figure 1B using MATa strains that carry plasmid-encoded a-factor-CaaX variants and plasmid-encoded HsRCE1 (Rce1) or ZMPSTE24 (Ste24) CaaX proteases. The source of the CaaX sequence is indicated below each specific sequence. The strain background for this experiment lacks endogenous a-factor (MFA1 and MFA2) and yeast CaaX protease genes (yWS164; MATa mfa1Δ mfa2Δ rce1Δ ste24Δ). MATa strains were mixed with MAT03b1 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. Plasmids are listed in Supplemental Table S3.