Fig. 1
Vacuolar membrane proteins are degraded in cells approaching stationary phase. a, b Integral vacuolar membrane proteins with diverse functions are degraded in cells approaching stationary phase. At time 0, log phase yeast cultures expressing the indicated GFP fusion proteins were diluted to OD600=0.2. Images and samples were collected at the indicated time points afterwards. a Fluorescent microscopy images showing the translocation of GFP signal from the surface to the lumen of vacuoles. DIC, differential interference contrast. Scale bar, 5 μm. b Immuno-blots showing the formation of free GFP bands. GFP-Pho8* presumably represents a truncated form. c Peripheral vacuolar membrane proteins do not translocate to vacuolar lumen in early stationary phase. Cells treated and images presented as in a. d Lysines at the cytosolic domain of Pho8 are dispensable for its turnover. The PHO8 locus was replaced with pho8 mutants carrying lysine-arginine substitutions as indicated. Cells treated and images presented as in a. e, f Turnover of GFP-Pho8 is dependent on vacuolar hydrolases. Cells were treated as in a. e Fluorescent microscopy images showing the appearance of uneven GFP signal in vacuoles in pep4Δ and atg15Δ cells. FM4-64 was used to label the vacuolar membrane. f Immuno-blots showing defective formation of free GFP in pep4Δ and atg15Δ cells