Figure 4. Autocrine Selection Produces Transporters with Increased S. cerevisiae a-Factor Transport.

(A) Four clones (A and B from the TM4–6 library and C and D from the TM10–12 library) were re-introduced into naive autocrine strains (that have not experienced FACS) to verify that increased autocrine signal is conferred by mutations in the clones. Each sample was measured by flow cytometry in biological triplicate populations (n > 25,000), and the normalized medians of the autocrine signal (black solid dots) and transporter expression (gray open diamonds) are plotted. The data were collected in high-throughput autocrine experiments (see STAR Methods) to make sure samples can be consistently compared. Common samples are duplicated in Figure S6A (right).

(B) Clones A, B, C, and D have reduced mating efficiency in Y. lipolytica MATA cells (equivalent to MATa mating type of S. cerevisiae) when substituting for YlSte6, suggesting a reduced ability to transport Y. lipolytica a-factor. The efficiency is the number of diploids for a sample relative to the mean number of diploids formed with WT YlSte6. The boxplots contain data from four different experiments, each with two biological replicates, with the box representing the middle 50%, median marked as a line, and the whiskers representing the outer quartiles of the distribution with individual outliers plotted as filled black diamonds.

(C) Sca-factor exported by populations expressing the indicated transporters was measured by an endpoint dilution biological assay in duplicate (crosses, average denoted by bars; units as in Figure 1C). Selected clones A, B, C, and D have increased a-factor export relative to WT YlSte6.

(D) Mating rescue of ste6D S. cerevisiae strains expressing transporter samples in biological triplicate plated on diploid selective media. Clones give increased rescue of S. cerevisiae mating compared with that of WT YlSte6.

See also Figure S4.