Saccharomyces cerevisiae divides asymmetrically to generate a rejuvenated daughter cell and an aged mother. Identifying the underlying asymmetric cellular components could uncover new aging factors and will inform general models for how cellular asymmetries are generated. A recent biochemical screen in our laboratory identified three proteins that are long-lived and asymmetrically retained for ~18 cell divisions in the plasma membrane of mother cells: Pma1, Mrh1, and Sur7. To uncover the mechanism underlying these proteins’ asymmetric localization, we have performed a high-throughput microscopy screen to discover genes that, when deleted, cause these asymmetric plasma membrane proteins to appear symmetric during budding.
We found that Pma1 asymmetry requires endocytic, vacuolar, and golgi-to-endosome trafficking whereas Mrh1 and Sur7 do not. Sur7 asymmetry is uniquely lost in a subset of cell wall and vesicular trafficking mutants (e.g., gas1Δ, ecm33Δ, and pho88Δ). A third set of mutants broadly affecting vesicle trafficking pathways (e.g., cdc50Δ, drs2Δ, rcy1Δ, and swa2Δ) accumulated cytoplasmic puncta containing Sur7, Mrh1, and Pma1 during budding, but only led to increased plasma membrane symmetry for Pma1. In contrast to Pma1 and Sur7, no mutants were identified that resulted in a strongly symmetric plasma membrane localization of Mrh1. Pulse-chase and photoconversion experiments have demonstrated that all identified mutants affect asymmetry at the step of new synthesis; that is, these mutants aberrantly express and localize new protein to the early bud at a time when, in wild-type, protein localization is repressed. A mechanistic analysis of these mutants and their role in plasma membrane protein asymmetry is ongoing and will be presented.