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A Vps21 endocytic module regulates autophagy.

Chen Y, Zhou F, Zou S, Yu S, Li S, Li D, Song J, Li H, He Z, Hu B, Björn LO, Lipatova Z, Liang Y, Xie Z, Segev N - Mol. Biol. Cell (2014)

Bottom Line: These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors.We propose that the endocytic Vps21 module also regulates autophagy.These findings support the idea that the two pathways leading to the lysosome--endocytosis and autophagy--converge through the Vps21 and Ypt7 GTPase modules.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.

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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.
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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.

Mentions: Vps9 is the GEF that activates Vps21 in endocytosis (Hama et al., 1999). To determine whether Vps9 also activates Vps21 in autophagy, we first determined whether vps9∆ mutant cells exhibit defects in autophagy. Assays described earlier showed that vps9∆ mutant cells exhibit defects in selective and nonselective autophagy similar to those observed for vps21∆ mutant cells (Figure 5, A–C, and Supplemental Figure S5, B and C). Moreover, fluorescence and electron microscopy analyses show that, like vps21∆ mutant cells, ∼40% of the vps9∆ mutant cells contain APCs outside their vacuoles under starvation (Figures 6A and 4, C and D, respectively). Remarkably, the autophagic defects displayed by vps9∆ mutant cells are very similar to those of vps21∆ mutant cells.


A Vps21 endocytic module regulates autophagy.

Chen Y, Zhou F, Zou S, Yu S, Li S, Li D, Song J, Li H, He Z, Hu B, Björn LO, Lipatova Z, Liang Y, Xie Z, Segev N - Mol. Biol. Cell (2014)

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.
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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.
Mentions: Vps9 is the GEF that activates Vps21 in endocytosis (Hama et al., 1999). To determine whether Vps9 also activates Vps21 in autophagy, we first determined whether vps9∆ mutant cells exhibit defects in autophagy. Assays described earlier showed that vps9∆ mutant cells exhibit defects in selective and nonselective autophagy similar to those observed for vps21∆ mutant cells (Figure 5, A–C, and Supplemental Figure S5, B and C). Moreover, fluorescence and electron microscopy analyses show that, like vps21∆ mutant cells, ∼40% of the vps9∆ mutant cells contain APCs outside their vacuoles under starvation (Figures 6A and 4, C and D, respectively). Remarkably, the autophagic defects displayed by vps9∆ mutant cells are very similar to those of vps21∆ mutant cells.

Bottom Line: These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors.We propose that the endocytic Vps21 module also regulates autophagy.These findings support the idea that the two pathways leading to the lysosome--endocytosis and autophagy--converge through the Vps21 and Ypt7 GTPase modules.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.

Show MeSH
Related in: MedlinePlus