Figure 7.

A vps34^ts mutant suppresses the partial missorting phenotypes of vac1 FYVE finger mutants. (A) DDY3477 (vps34^ts) displays temperature-sensitive PtdIns(3) kinase activity. Wild-type yeast and DDY3477 yeast expressing wild-type VAC1, C221S-vac1, or C237S-vac1 were grown for 16 h at 25 or 30°C in minimal media containing [³H]myo-inositol. A 0.1 vol of YPD was added, and incubations were continued for 2.5 h. One 30°C culture was shifted to 38.5°C for the last 30 min of the 2.5-h incubation. Deacylated lipid extracts were prepared from lysates of these cultures and separated by HPLC. This procedure allowed the complete separation of the deacylated glycerophosphoinositols gPI(3)P and gPI(4)P. For each strain, the amount of gPI(3)P that was produced is expressed relative to the amount of gPI(4)P, whose level remained largely unchanged at 25, 30, and 38.5°C. (B) DDY3477 (vps34^ts) displayed a temperature-sensitive vacuolar protein sorting defect. The ability of the vps34^ts strain DDY3477 to sort CPY was determined as described in Figure 4 and MATERIALS AND METHODS, except that cultures were incubated at 25 or 38.5°C for 10 min before labeling at those temperatures. (C) The abilities of vac1 FYVE finger mutants to sort CPY to the vacuole were compared in wild-type and vps34^ts backgrounds at the semipermissive temperature of 30°C. GTY104 (Δvac1) expressing no Vac1p (lane 1), VAC1 (lane 3), C221S-vac1 (lane 4), and C237S-vac1 (lane 6) and GTY110 (Δvac1vps34^ts) expressing VAC1 (lane 2), C221S-vac1 (lane 5), and C237S-vac1 (lane 7) were pulse labeled with [³⁵S]ProMix for 10 min and chased with unlabeled amino acids for 30 min at 30°C. CPY was immunoprecipitated from the combined intracellular and extracellular fractions of lysates generated from these cells as described in Figure 4 and MATERIALS AND METHODS.