FIGURE 3.
PI(4,5)P2 regulation by eisosomes inhibits Pmk1 activation. A, control or its3-1 mutant cells were incubated at 30 °C and assayed for Pmk1 activation at the indicated time points. B, cells carrying the pREP41x-its3+ plasmid were grown under conditions to repress (+B1) or induce (−B1) overexpression of Its3 and then tested for Pmk1 activation at the indicated time points after the addition of 1 m KCl. C and D, the indicated strains were tested for Pmk1 activation at the indicated time points after the addition of 1 m KCl. All strains carry a HA6H-tagged chromosomal version of pmk1+. For all panels cells were harvested, and Pmk1-HA6H was affinity-purified. Each sample was then immunoblotted for total Pmk1 with anti-HA and for activated Pmk1 with anti-phospho-p42/44 antibodies. R.U. indicates relative units of activated versus total Pmk1. All experiments were performed in triplicate, and graphs show mean relative units ± S.D. p values were determined by unpaired Student's t test.