A Rab5 GTPase module is important for autophagosome closure

Abstract

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Down syndrome fibroblasts exhibit diminished autophagic clearance and endosomal dysfunction after serum starvation

“…The observation of disrupted endosomal pathways in DS would be consistent with previous reports of enlarged early endosomes and increased endocytic uptake in Tri21 models (71,72,112,113). Rab5 is critical for endocytosis, endosomal sorting (74) and it also participates in autophagosome formation and closure (73,(114)(115)(116)(117)(118)(119) demonstrates fusion of autophagosomes with late endosomes. As mentioned previously, late endosomes have the ability to fuse directly with the lysosome (80); thus, increased autophagosome-late endosome co-localization might increase the quantity of cargo destined for lysosomal degradation and possibly delay autophagic clearance.…”

“…The early endosome serves as sorting station for endocytosed cargo and is the first organelle of the endocytic 22 machinery to receive incoming material. Rab5, a prominent member of the Ras GTPase family, resides in early endosomal vesicles (70,73) and is involved in endocytosis and endosomal sorting (74) In order to examine possible interaction between autophagic vesicles and early endosome function in DS, we investigated the effect of SS on Rab5 protein abundance in DS fibroblasts relative to euploid controls. Basal abundance of Rab5 was observed to be similar between CTL and DS fibroblasts ( Figure 5A&B).…”

TRAPPC11 functions in autophagy by recruiting ATG2B‐WIPI4/WDR45 to preautophagosomal membranes

“…This assay faithfully reports the state of the isolation membranes in both mammalian and lower eukaryotic systems. 47,49,50 Following depletion, HeLa cells were FIGURE 1 Depletion of TRAPPC11 affects autophagic flux. (A) HeLa cells expressing mRFP-GFP-LC3 were subjected to a knockdown (KD) with either non-specific (NS) small interfering (si)RNA, or siRNA targeting TRAPPC2, TRAPPC8, TRAPPC11 or TRAPPC12.…”

Vrl1 relies on its VPS9‐domain to play a role in autophagy in Saccharomyces cerevisiae

“…Rab5 GEFs are required for the localization of PI3P, which plays important roles in both vesicle trafficking and autophagy ( Lindmo and Stenmark, ; Dall’Armi et al, ; Lipatova et al, ). PI3P was largely mislocalized from endosomes to the vacuole rim or autophagosomes in Vps21 or its GEF mutants (Nickerson et al, ; Bean et al, ; Zhou et al, ). Vrl1, but not Vrl1 D373A , was partially able to relocate PI3P from the vacuole rim to endosomes under normal growth conditions as probed with GFP‐FYVE (Fab1, YOTB, Vac1, and EEA1) (Bean et al, ).…”

“…However, GFP-Vrl1 significantly co-localized with mCherry-Atg8 in mutants with defects in the late autophagy under starvation conditions. Specifically, GFP-Vrl1 co-localized with mCherry-Atg8 marked unclosed autophagosome clusters at the vacuolar rim in the pep12Δ mutant (Figure 2A; Chen et al, 2014;Zhou et al, 2017); with mCherry-Atg8 marked closed autophagosomes dispersed in the cytosol in the ypt7Δ mutant (Kirisako et al, 1999), and with mCherry-Atg8 marked autophagic bodies in vacuoles in the pep4 mutant (Takeshige et al, 1992) ( Figures 5A and B). As expected, we did not find colocalization for GFP-Vrl1 and mCherry-Atg8 in atg1Δ under starvation conditions ( Figure 5A), during which autophagy was blocked at the early step and Atg8 mainly gathered on pre-autophagosomal structures (PAS) (Cheong et al, 2008).…”