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. 2018 Nov 1;29(22):2751–2765. doi: 10.1091/mbc.E18-03-0158

FIGURE 5:

FIGURE 5:

Characterization of Par32 and its various mutants in response to rapamycin exposure. (A) Δpar32 cells expressing Par32 4x mut do not recover from exposure to rapamycin. Exponentially growing W303A or Δpar32 cells (OD600 0.6–0.8) expressing the indicated PAR32 construct on a cen/ars plasmid were treated with rapamycin (200 ng/ml in YPD) for 5 h at 30°C. Cells were then washed and plated on YPD. Cells were imaged after incubation for 2 d at 30°C. The leftmost spot in each case corresponds to 2 μl of a culture with OD600 0.5. Spots to the right of this correspond to 2 μl of sequential fivefold dilutions. (B) Nuclear targeting of Par32 4x mut does not rescue growth after rapamycin exposure. W303A or Δpar32 cells expressing the indicated constructs (NLS, SV40 Large T Antigen nuclear localization sequence) were, where indicated, untreated or treated with rapamycin (200 ng/ml in YPD) for 5 h at 30°C prior to washing and plating on YPD. Plates were imaged after 2 d at 30°C. (C) Deletion of Mep1 and Mep3 rescues the Δpar32 rapamycin-sensitive growth phenotype. The indicated strains were treated with rapamycin and plated as in A. (D) The sensitivity of cells lacking Par32 is ammonium dependent. The experiment was performed as in C but cells were grown overnight in SD–ammonium and plated on either SC or SD–ammonium.