Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt.

Péter Lőrincz,Lili Anna Kenéz,Sarolta Tóth,Zsófia Simon-Vecsei,Viktória Kiss,Ágnes Varga,Tamás Csizmadia,Gábor Juhász

doi:10.7554/elife.45631

Abstract

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lőrincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.

Highlights

Lysosomal degradation is essential for the survival and homeostasis of eukaryotic cells
As Vps8 and Vps41 are suggested to occupy the same binding site in CORVET and HOPS, respectively (Peplowska et al, 2007), we tested whether the overexpression of a complex specific subunit may result in the inhibition of the other complex
We found that – similar to vps11 or vps39 RNAi cells - Vps8 overproducing cells are swollen and contain enlarged late endosomes based on both confocal and electron microscopy (Figure 1A–D,F–I, Figure 1—figure supplement 1). This phenotype resembles to the yeast data: Vps8 overexpression delayed late endosome maturation and caused the accumulation of multivesicular bodies (MVBs) proximal to the vacuole (Markgraf et al, 2009) and the overproduction of Vps3 caused vacuole fragmentation (Peplowska et al, 2007), which is a definitive characteristic of HOPS loss-of function in yeast cells (Raymond et al, 1992)

Summary

Introduction

Lysosomal degradation is essential for the survival and homeostasis of eukaryotic cells. HOPS was identified in yeast and is defined by two Ypt (Rab in higher eukaryotes) binding subunits Vps and Vps on its opposing ends (Balderhaar et al, 2013; Balderhaar and Ungermann, 2013; Brocker et al, 2012; Lurick et al, 2018; Peplowska et al, 2007; Plemel et al, 2011; Rieder and Emr, 1997; Seals et al, 2000; Wickner and Schekman, 2008; Wurmser et al, 2000). A closely related multisubunit complex termed CORVET (Class C core endosome vacuole tethering) mediates the tethering and fusions of Vps (Rab in higher eukaryotes) positive membranes

Methods

Results

Conclusion