1. Low pH alters Ras1 localization and its activation state, but these changes are not responsible for the effect of pH on morphology.
Low pH alters Ras1 localization and its activation state, but these changes are not responsible for the effect of pH on morphology. (A) Total Ras levels (top) were comparable at pH 4 and 7, although cleavage prevailed at pH 4. (Top) Western blot results for the GTP-bound fraction of the lysates. (Middle) Anti-Ras1 Western blot results with whole-cell lysates. (Bottom) Gelcode Blue stain of the membrane blotted in the top panel, to indicate loading. Cells were grown for 3 h in YNBNC at the indicated pH. (B) Western blot of the Ras1 profile and microscopy of tetO-UME6 and tetO-NRG1 strains with and without 50 µg per ml doxycycline. (C) Ras1 profile of cells of the indicated genotype grown for 3 h in YNB–GlcNAc–0.2% glucose (YNBNG) adjusted to pH 7 and subsequently buffered to the indicated pH with citrate for the indicated time. (D) Ras1-GTP binding of RAS1 cells grown for 3 h in YNBNG adjusted to pH 7 and subsequently buffered to the indicated pH with citrate for 30 min. (E) Quantitative morphology of the indicated strains grown for 3 h in YNBNC at 37°C at pH 4 or pH 7. Error bars represent one standard deviation.
Jeffrey M. Hollomon et al. mSphere 2016; doi: /mSphere