Figure 2.

The Effect of Vps1 Mutations on Functions Requiring Vps1

(A) Whole-cell extracts from yeast expressing mutated Vps1, separated by SDS-PAGE, blotted, and probed with anti-Vps1 antibodies. Apart from KRR-EEE, all mutants expressed at normal levels. Loading control is Ponceau-stained blot.

(B) The effect of mutations on growth at a range of temperatures for cells expressing integrated vps1 mutants. Shown is growth at 37°C in the presence and absence of the osmotic support molecule sorbitol.

(C) Vps1 is required for normal vacuolar morphology, recycling of the snare Snc1 through endosomes, and for peroxisomal fission. The effect of mutations on these functions was assessed using FM4-64, GFP-Snc1, and GFP-PTS1, respectively, and representative data are shown in upper, middle, and lower panels.

(D) Carboxypeptidase Y normally functions in the vacuole where it is cleaved to its mature form (mCPY). In the absence of vps1, and in the KRR and K473E mutants, some of this material accumulates in a precursor form (pCPY). Below is a GAPDH loading control.

(E) Uptake of the fluid phase marker lucifer yellow was analyzed after incubation at 21°C for up to 90 min. Localization of the majority of the dye to vacuoles (large round or lobed structure) was counted. n = 100 cells in each of two independent experiments. Errors are SD.

(F) Images of cells at the 60 min time point with lucifer yellow.