A Vps21 endocytic module regulates autophagy.
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.
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
Mentions: To gain insight into the autophagic step blocked in vps21∆ mutant cells under starvation, we determined the vacuolar morphology and localization of GFP-Atg8 by live-cell fluorescence microscopy. GFP-Atg8 that resides on the inner membrane of APs is delivered to the vacuole after the AP and vacuole fuse, where it is degraded (Kirisako et al., 1999; Huang et al., 2000). When grown in rich medium (YPD), GFP-Atg8 is diffuse, with an occasional dot near the vacuole of both wild-type and vps21∆ mutant cells. Under starvation (synthetic defined medium that lacks nitrogen and amino acid [SD-N]), wild-type cells generally contain one or two dots per cell of GFP-Atg8 near the vacuole representing the AP, and a substantial amount is delivered to the vacuole for degradation (as indicated by GFP fluorescence observed inside the vacuoles). In vps21∆ mutant cells starved for nitrogen, some cells contain green fluorescence in their vacuoles (Figure 2A), which is in agreement with the ∼50% processed GFP found in these cells (Figure 1B). Of importance, after 4 h of nitrogen starvation, ∼45% of the vps21∆ mutant cells accumulate GFP-Atg8 in crescent-like structures near the vacuole (stained by FM4-64). This GFP-Atg8 accumulation phenotype of vps21∆ mutant cells occurs only under starvation and can be complemented by expression of Vps21 from a plasmid (Figure 2A). A similar phenotype was observed in vps21ts mutant cells at 37°C (Supplemental Figure S1B). Analysis of the two other Rab5-related genes, YPT52 and YPT53, revealed that in cells deleted for these genes, the GFP-Atg8 pattern is similar to that seen in wild- type cells (Supplemental Figure S2D).
Affiliation: College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.