FIGURE 5:

Autophagic phenotypes of vps9∆ mutant cells can be suppressed by active Vps21. (A) vps9∆ mutant cells are defective in nonselective autophagy measured by ALP activity. ALP activity was determined as described in Figure 1A in wild-type, atg1∆ (as a negative control), and vps9∆ mutant cells. Vps9∆ mutant cells exhibit a 75% defect (p < 0.0005). (B) The GFP-Atg8 processing defect of vps9∆ mutant cells is suppressed by expression of the Vps21 wild-type or the putative GTP-locked mutant proteins but not the putative GDP-locked mutant protein. GFP-Atg8 processing was determined as described in Figure 1B in wild-type and vps9∆ mutant cells transformed with empty plasmid (ø) or plasmids for overexpression of wild-type Vps21 (Vps21-WT), Vps21-S21N (putative GDP-locked Vps21 mutant protein), or Vps21-Q66L (putative GTP-locked Vps21 mutant). Vps9∆ mutant cells (ø or putative GDP-locked Vps21) exhibit a defect in GFP-Atg8 processing compared with wild-type cells (p < 0.002). (C) The Ape1 processing defect of vps9∆ mutant cells is partially suppressed by expression of wild type or the putative GTP-locked Vps21 mutant, but not the putative GDP-locked mutant. Ape1 processing was determined (as in Figure 1C, top) in wild-type and vps9∆ mutant cells transformed with plasmids for Vps21 expression (as in B). Vps9∆ mutant cells (ø or putative Vps21-GDP locked) exhibit a defect in Ape1 processing compared with wild-type cells (p < 0.005). (D) The growth defect of vps9∆ mutant cells is suppressed by expression of wild-type or GTP-locked, but not the putative GDP-locked, Vps21. The growth of wild-type and vps9∆ mutant cells transformed with plasmids for Vps21 expression (as in B) was determined on SD plates at 26 and 37°C (1/10 serial dilution from left to right). Vps9∆ mutant cells (ø or the putative GDP-locked Vps21) exhibit a growth defect at 37°C when compared with wild-type cells or vps9∆ mutant cells expressing Vps21-WT or Vps21-GTP. Error bars and ± represent STD. Results represent three independent experiments.