Figure 2. Alanine-scanning mutagenesis of the PRLYL motif and cell fractionation to localize Vps10p, Vps29p and Vps35p.

(A) CPY sorting of vps35Δ cells expressing the PRLYL alanine-scanning mutants. vps35Δ cells were transformed with centromeric plasmids (low copy number) to express wild-type VPS35, the L99P mutant or the PRLYL motif mutants. Cells were pulse-labelled with ³⁵S-methionine for 10 min and chased for 30 min before the culture was transferred to ice and precipitated with 10% TCA. After lysis, total CPY was recovered by immunoprecipitation. The L99P and R98A mutants both fail to complement the CPY sorting defect in vps35Δ yeast. In the lower panel, Vps35p was recovered by immunoprecipitation showing that all the mutants are equally expressed. (B) Cells were labelled with ³⁵S-methionine before being fractionated into P13 (vacuolar membrane), S100 (cytosolic) and P100 (Golgi, endosomes and small vesicles) fractions to determine the distribution of Vps10p, Vps29p and Vps35p. Loss of retromer function results in Vps10p being mislocalized to the vacuolar membrane (P13) fraction and a similar phenotype is produced in cells expressing the L99P mutant. When the levels of Vps35p in each fraction is quantified, it is apparent that the behaviour of Vps35(L99P) protein is similar to Vps35p in vps26Δ cells. (C) Wild-type cells (MSY10-21) expressing either wild-type VPS35 or the vps35(L99P) mutant were fractionated as above and Vps10p and Vps26p were recovered by immunoprecipitation. In a wild-type background there is little discernable shift of Vps10p to the vacuolar (P13) fraction due to the L99P mutation.