Abstract

The mammalian homotypic fusion and vacuole protein sorting (HOPS) complex is comprised of six subunits: VPS11, VPS16, VPS18, VPS39, VPS41 and the Sec1/Munc18 (SM) family member VPS33A. Human HOPS has been predicted to be a tethering complex required for fusion of intracellular compartments with lysosomes, but it remains unclear whether all HOPS subunits are required. We showed that the whole HOPS complex is required for fusion of endosomes with lysosomes by monitoring the delivery of endocytosed fluorescent dextran to lysosomes in cells depleted of individual HOPS proteins. We used the crystal structure of the VPS16/VPS33A complex to design VPS16 and VPS33A mutants that no longer bind each other and showed that, unlike the wild‐type proteins, these mutants no longer rescue lysosome fusion with endosomes or autophagosomes in cells depleted of the endogenous proteins. There was no effect of depleting either VIPAR or VPS33B, paralogs of VPS16 and VPS33A, on fusion of lysosomes with either endosomes or autophagosomes and immunoprecipitation showed that they form a complex distinct from HOPS. Our data demonstrate the necessity of recruiting the SM protein VPS33A to HOPS via its interaction with VPS16 and that HOPS proteins, but not VIPAR or VPS33B, are essential for fusion of endosomes or autophagosomes with lysosomes.

Highlights

  • In mammalian cells, the transport of macromolecules to the lysosome depends on tightly regulated multi-step processes involving bi-directional vesicular transport and membrane fusion between distinct organelles

  • Recruitment of VPS33A to the homotypic fusion and vacuole protein sorting (HOPS) complex via interaction with VPS16 is required for endosome–lysosome fusion To test whether the interaction of VPS33A and the rest of HOPS is required for endosome–lysosome fusion, we mutated in VPS16 the critical residues alanine 669 (A669D) and/or arginine 725 (R725E), which we have previously shown to be required for binding of VPS16 to VPS33A without substantially altering folding or stability [(44), Figure 2A]. small interfering RNA (siRNA)-resistant HA-tagged wild-type HA-VPS16(WT) or mutant HA-VPS16(A669D), HAVPS16(R725E), or HA-VPS16(A669D/R725E) were stably expressed in HeLaM cells

  • We investigated the requirement for HOPS proteins in the final stages of delivering endocytosed cargo from endosomes to lysosomes and the fusion of autophagosomes with lysosomes in mammalian cells

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Summary

Introduction

The transport of macromolecules to the lysosome depends on tightly regulated multi-step processes involving bi-directional vesicular transport and membrane fusion between distinct organelles. Mammalian cells take up cell-surface membrane proteins, bound ligands, lipids and extracellular fluid into vesicles that subsequently fuse with early endosomes. These mature into late endosomes ( known as multivesicular bodies) that fuse with lysosomes to form endolysosomes [1,2]. There is evidence that the mammalian HOPS protein VPS41 has additional functions that are independent of the complex in the fusion of lysosome-associated membrane protein (LAMP) carriers with late endosomes [30], and the biogenesis of regulated secretory granules [31]. We found that neither VPS33B nor VIPAR are required for these fusion events and are physically neither part of the human HOPS complex nor of the human CORVET complex

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