A Vps21 endocytic module regulates autophagy.
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.
- rab GTP-Binding Proteins/metabolism*
- Biological Transport
- Guanine Nucleotide Exchange Factors/metabolism
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Figure 8: Vps21 colocalizes with autophagosomal markers. (A) Colocalization of GFP-Vps21 with RFP-Ape1 in wild-type and pep12∆ mutant cells. GFP-Vps21 and RFP-Ape1 were integrated into the genome of wild-type (top) and pep12∆ mutant (bottom) cells. Cells grown in SD or shifted to SD-N were visualized by live-cell fluorescence microscopy. Left to right, GFP-Vps21, RFP-Ape1, merge, and percentage of cells with colocalization. In wild-type cells, Ape1 is found inside the vacuole (Shintani et al., 2002) and localizes to a single dot of the AP. In pep12∆ mutant cells, Ape1 accumulates outside the vacuole in APs. Vps21 accumulates in multiple dots or a cluster per cell, and in 14–26% of the cells, one of the Vps21 dots colocalizes with Ape1. (B) Colocalization of RFP-Vps21 with GFP-Atg8 in wild-type and pep12∆ mutant cells. GFP-Atg8 was integrated into the genome of wild-type (top) and pep12∆ mutant (bottom) cells, and RFP-Vps21 was expressed from a plasmid (Markgraf et al., 2009). The experiment was performed as described in A. Left to right, RFP-Vps21, GFP-Atg8, merge, and percentage of cells with colocalization. In wild-type cells, Atg8 is found inside the vacuole and localizes to a single dot of the AP. In pep12∆ mutant cells, Atg8 accumulates outside the vacuole, in SD medium to a single dot, and in SD-N to a cluster (as in vps21∆ mutant cells). RFP-Vps21 localizes to multiple dots or a cluster, and one of them colocalizes with GFP-Atg8 in ∼14 and ∼43% of wild-type and pep12∆ mutant cells, respectively. Percentage of cells with colocalization of the Vps21 with the AP marker was determined in cells that contain both green and red puncta; >130 wild-type cells; >350 pep12∆ mutant cells. Arrows point to areas of colocalization of Vps21 with the AP marker; bar, 5 μm; ± represents SD. Results in A represent three independent experiments and in B four independent experiments. (C) Convergence of the endocytic and autophagic pathways. We propose that the two pathways leading to the lysosome converge through two Ypt/Rab modules: Vps21 (black) and Ypt7 (gray). Ypt7, its GEF, and effectors were previously shown to regulate both endocytosis and autophagy (Noda et al., 2009). A role for Vps21, its GEF, and effectors was established in endocytosis (Stack et al., 1995; Epp et al., 2011). Here we show that the endocytic Vps21 module also regulates autophagy. Factors that function downstream of Vps21 include a number of effectors, the tethering complex CORVET (Vps3 and Vps8), the adaptor/tether Vac1, and the SM protein Vps45, as well as the SNARE Pep12.
Appearance of Ape1 and Atg8 in a single AP dot occurs in a fraction of wild-type cells. In ∼15% of wild-type cells with an AP dot, Vps21 colocalizes with these autophagosomal markers (Figure 8, A and B). The relatively low colocalization of Vps21 with AP markers can be attributed to the fact that Vps21 is present on APs transiently. If true, an increased level of colocalization should be seen in cells that accumulate APs, especially if the Vps21-mediated step is blocked. Therefore we tested the colocalization of Vps21 with Ape1 and Atg8 in pep12∆ mutant cells. Because the Pep12 functions as a SNARE in the Vps21-mediated step, the Vps21-mediated step is blocked in pep12∆ mutant cells. An increase of colocalization of Vps21 with Ape1 is seen in pep12∆ mutant cells (Figure 8A). Moreover, accumulation of GFP-Atg8 crescents in >70% of pep12∆ mutant cells (Figure 7C) facilitates observation of the colocalization of Vps21 with GFP-Atg8. Indeed, under starvation, Vps21 colocalizes with dots or crescents of GFP-Atg8 in >40% of the pep12∆ mutant cells (Figure 8B). The colocalization of Vps21 with two autophagosomal markers supports the idea that Vps21 plays a role in autophagy.