FIGURE 6:

Accumulation of GFP-Atg8 crescents in vps9∆ mutant cells can be suppressed by active Vps21. (A) Vps9 suppresses accumulation of GFP-Atg8 crescents in vps9∆ mutant cells. GFP-Atg8 was integrated into the genome of wild-type and vps9∆ mutant cells. Cells transformed with a 2 μ plasmid for overexpression of Vps9 (or empty plasmid [ø] as a negative control) were analyzed for GFP-Atg8 pattern and vacuolar morphology as described for Figure 2. About 40% of the vps9∆ mutant cells accumulate GFP-Atg8 in crescents that overlap with the vacuole rim, and this phenotype is suppressed by overexpression of Vps9 from a plasmid (number of cells visualized for each strain, >450). (B) Active Vps21 partially suppresses the accumulation of GFP-Atg8 crescents in vps9∆ mutant cells. The experiment was done as in A, except that vps9∆ mutant cells were transformed with plasmids for overexpression of the different nucleotide-locked forms of Vps21 (as in Figure 5, B–D). Fewer vps9∆ mutant cells accumulate GFP-Atg8 crescents when Vps21-WT and the GTP-locked Vps21 mutant protein, but not the putative GDP-locked Vps21, are overexpressed (<20% and >40%, respectively; number of cells visualized for each strain, >650). Arrows point to GFP-Atg8 crescents; bar, 2 μm; ± represents STD. Results represent two independent experiments.