Assaying VPS8D knockdown phenotypes
To measure survival rates after hypoosmotic shock, wildtype andVPS8D -KD strains (3 of each, with independent isolates of the mutant strain) were grown to log phase in 10 ml SPP. Cell densities were measured and equalized, and 0.5ml aliquots were diluted 1:100 in 50 ml SPP medium with or without 1 µg/ml CdCl2. Cells were grown for 16 hours at 30°C with agitation. Culture densities were measured, and then cells were pelleted by centrifugation in a clinical centrifuge for 1 minute and resuspended gently in 10 mM Tris-HCl buffer, pH 7.4. After an overnight incubation, culture densities were once again measured to calculate survival rates. For experiments monitoring changes in CVC markers, the VPS8D -KD strains endogenously expressing Dop1p-mNeon, Vma4p-mNeon or Scr7p-mNeon were grown as above in non-inducing or inducing conditions. Cells were withdrawn and fixed for imaging after 3, 5 and 8 (Fig. 2) or 16h (Fig. 3).