Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes

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.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…We observed extensive interactions between VPS11, VPS16, VPS8, TGFBRAP1, and VPS33A. In contrast, VIPAS39 and VPS33B interacted with each other but not with any of the other canonical CORVET/HOPS subunits in line with recent data (12).…”

“…We confirm that TGFBRAP1 and VPS8 are the mammalian CORVET-specific subunits (with TGFBRAP1 as the yeast vps3 ortholog) (11,35). In contrast to previous data (16), however, our data indicate that VIPAS39 and VPS33B, two CORVET/HOPS subunit homologues not present in yeast, are not part of CORVET/HOPS as also suggested by others (12,21,36 interactions of VPS33B and/or VIPAS39 with CORVET/HOPS or the existence of VPS33B-VIPAS39-HOPS interactions in other cellular systems. However, the existence of a VPS33/ VIPAS39 complex independent of other core subunits might explain why only mutations in VPS33B and VIPAS39 but not any of other HOPS subunits are associated to ARC syndrome (21)(22)(23)33) and why all HOPS complex subunits, but not VPS33B and VIPAS39, are required for Ebola-virus infection (19).…”

“…Here we provide an extensive map of the mammalian CORVET, HOPS, and VPS33B-VIPAS39 complexes describing their intersubunit interactions and membrane recruitment. In agreement with Wartosch et al (12) we find that VIPAS39 and VPS33B are not part of the CORVET or HOPS complex but assemble in a distinct complex. We find that the CORVET complex is prevented from recruitment to LE (in line with its function at the EE).…”

“…Published data regarding the role of VPS33B and VIPAS39 as members of the HOPS complex are seemingly conflicting (12,16). Our endogenous IP data (Fig.…”

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis

“…We observed a statistically significant, widespread suppression in nearly 90% of all autophagy-associated genes tested (93% of FOXO targets, 83% of TFEB targets), with the single exception of Sqstm1, which was upregulated in mutant cells ( Figure 4H). Among the significantly downregulated genes were those encoding autophagic machinery components linked to: autophagic vacuole formation (Atg5, Atg12, Atg16l1, Gabarap Gabarapl1, Gabarapl2, Becn1); targeting proteins to the autophagosome (Atg4b); linking autophagosomes to lysosomes (Vps33a, Vps11); protein transport (Atg10, Atg16l2); and protein ubiquitination (Atg3) (56,57). Genes associated with transmitting an autophagic response to intracellular signals (Pik3c3, processes further.…”