FIGURE 3: The effect of Vps1 mutations on functions requiring Vps1. (A) Whole cell extracts were made from yeast expressing mutated vps1 as the only form of Vps1. These were separated by SDS-PAGE, transferred to PVDF membrane and probed with anti-CPY antibodies. Two bands are observed, the mature protein (mCPY) and the immature, precursor form (pCPY). (B) The lipophylic dye FM4-64 was used to determine whether the mutations affect vacuolar morphology. (C) Cells were transformed with a peroxisomal targeting sequence fused to GFP. This allowed the morphology of peroxisomes to be visualized in strains lacking vps1 and dnm1 but re-transformed with VPS1 wild type or mutants. (D) A strain was generated expressing GFP-Snc1-SUC2 that allows endosomal cycling to be investigated. GFP indicates localization of this reporter. (E) The same GFP-Snc1-SUC2 reporter also allows invertase activity present at the cell surface to be detected in a colorimetric assay on plates as described in text. (F) Endocytic uptake into cells was monitored using Lucifer yellow which was incubated for 90 min with all strains. The predominant localization in 100 cells of each strain, in 3 independent repeats was counted. Error bars are standard error of the mean.

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