The multisubunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for late endosome-lysosome and autophagosome-lysosome fusion in mammals. We have determined the crystal structure of the human HOPS subunit Vps33A, confirming its identity as a Sec1/Munc18 family member. We show that HOPS subunit Vps16 recruits Vps33A to the human HOPS complex and that residues 642-736 are necessary and sufficient for this interaction, and we present the crystal structure of Vps33A in complex with . Mutations at the binding interface disrupt the Vps33A-Vps16 interaction both in vitro and in cells, preventing recruitment of Vps33A to the HOPS complex. The Vps33A-Vps16 complex provides a structural framework for studying the association between Sec1/Munc18 proteins and tethering complexes. E ukaryotic cells tightly regulate the movement of macromolecules between their membrane-bound compartments. Multiple proteins and protein complexes interact to identify vesicles or organelles destined to fuse, bring them into close proximity, and then fuse their membranes, thereby allowing their contents to mix (1). Multisubunit tethering complexes modulate key steps in these fusion events by recognizing specific Rab-family small GTPases on the membrane surfaces, physically docking the membranes and then recruiting the machinery that effects the membrane fusion (2, 3).In metazoans, the multisubunit tethering complex homologous to the yeast homotypic fusion and vacuole protein sorting (HOPS) complex (4-7) is required for the maturation of endosomes (8); the delivery of cargo to lysosomes (9) and lysosomerelated organelles, such as pigment granules in Drosophila melanogaster (10); and the fusion of autophagosomes with late endosomes/lysosomes (11). The mammalian HOPS complex comprises six subunits (Vps11, Vps16, Vps18, Vps33A, Vps39, and Vps41) (4-6). Homologs of HOPS components can be identified in almost all eukaryotic genomes (12) and are thought to be essential; for example, removal of the Vps33A homolog carnation (car) in Drosophila is lethal during larval development (13).HOPS components have been identified in animal models of human disease. A missense point mutation in the murine Vps33a gene gives rise to the buff mouse phenotype, characterized by pigmentation, platelet activity, and motor deficiencies (14). This phenotype closely resembles the clinical presentation of Hermansky-Pudlak syndrome (HPS) (15), and a mutation in the human VPS33A gene has been observed in a patient with HPS who lacked mutations at other known HPS loci (14). In metazoans, there is a second homolog of yeast Vps33 called Vps33B, but disruption of the VPS33B gene in humans gives rise to a clinical phenotype distinct from HPS (16).Human Vps33A is predicted to be a member of the Sec1/ Munc18 (SM) family of proteins (7, 17) that, together with SNAREs, comprise the core machinery essential for membrane fusion in eukaryotes (18). Three SNAREs with glutamine residues at the center of their SNARE domain (Q a -, Q b -, and Q c -SNARE...

The vast expansion in recent years of the cellular processes promoted by the endosomal sorting complex required for transport (ESCRT) machinery has reinforced its identity as a modular system that uses multiple adaptors to recruit the core membrane remodelling activity at different intracellular sites and facilitate membrane scission. Functional connections to processes such as the aurora B-dependent abscission checkpoint also highlight the importance of the spatiotemporal regulation of the ESCRT machinery. Here, we summarise the role of ESCRTs in viral budding, and what we have learned about the ESCRT pathway from studying this process. These advances are discussed in the context of areas of cell biology that have been transformed by research in the ESCRT field, including cytokinetic abscission, nuclear envelope resealing and plasma membrane repair.

Background: Understanding of the true asymptomatic rate of infection of SARS-CoV-2 is currently limited, as is understanding of the population-based seroprevalence after the first wave of COVID-19 within the UK. The majority of data thus far come from hospitalised patients, with little focus on general population cases, or their symptoms. Methods: We undertook enzyme linked immunosorbent assay characterisation of IgM and IgG responses against SARS-CoV-2 spike glycoprotein and nucleocapsid protein of 431 unselected general-population participants of the TwinsUK cohort from SouthEast England, aged 19-86 (median age 48; 85% female). 382 participants completed prospective logging of 14 COVID-19 related symptoms via the COVID Symptom Study App, allowing consideration of serology alongside individual symptoms, and a predictive algorithm for estimated COVID-19 previously modelled on PCR positive individuals from a dataset of over 2 million. Findings: We demonstrated a seroprevalence of 12% (51 participants of 431). Of 48 seropositive individuals with full symptom data, nine (19%) were fully asymptomatic, and 16 (27%) were asymptomatic for core COVID-19 symptoms: fever, cough or anosmia. Specificity of anosmia for seropositivity was 95%, compared to 88% for fever cough and anosmia combined. 34 individuals in the cohort were predicted to be Covid-19 positive using the App algorithm, and of those, 18 (52%) were seropositive. Interpretation: Seroprevalence amongst adults from London and SouthEast England was 12%, and 19% of seropositive individuals with prospective symptom logging were fully asymptomatic throughout the study. Anosmia demonstrated the highest symptom specificity for SARS-CoV-2 antibody response.

The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/169185 doi: bioRxiv preprint first posted online 2 ABSTRACT Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion.CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.