Homotypic Fusion And Protein Sorting Research Articles

A condensates-to-VPS41-associated phagic vacuoles conversion pathway controls autophagy degradation in plants

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.

Hypomyelinated vps16 Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.

Myelin basic protein antagonizes the SARS-CoV-2 protein ORF3a-induced autophagy inhibition

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS.

HOPS-Dependent Endosomal Escape Demands Protein Unfolding.

The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the translocation of a protein involves two discrete steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules can achieve high protein uptake, but few are capable of efficient endosomal escape. One exception to this rule is ZF5.3, a mini-protein that exploits elements of the natural endosomal maturation machinery to translocate across endosomal membranes. Although some ZF5.3-protein conjugates are delivered efficiently to the cytosol or nucleus, overall delivery efficiency varies widely for different cargoes with no obvious design rules. Here we show that delivery efficiency depends on the ability of the cargo to unfold. Using fluorescence correlation spectroscopy, a single-molecule technique that precisely measures intracytosolic protein concentration, we show that regardless of size and pI, low-Tm cargoes of ZF5.3 (including intrinsically disordered domains) bias endosomal escape toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal, even if the Tm is high. These findings imply a novel pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.

HOPS-dependent lysosomal fusion controls Rab19 availability for ciliogenesis in polarized epithelial cells.

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering homotypic fusion and protein sorting (HOPS) complex disrupts this actin clearing and ciliogenesis, but it remains unclear how the ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body, and that this effect is specific to polarized epithelial cells. We also find that Rab19 functions in endolysosomal cargo trafficking in addition to having its previously identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion leads to the abnormal accumulation of Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin clearing and ciliogenesis in polarized epithelial cells.

Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing.

Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. Autophagic activity gradually declines with age due to genetic control, and this change contributes to the accumulation of cellular damage at advanced ages, thereby causing cells to lose their functionality and viability. This could be particularly problematic in post-mitotic cells including neurons, the mass destruction of which leads to various neurodegenerative diseases. Here, we aim to uncover new regulatory points where autophagy could be specifically activated and test these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For our experiments, we used Drosophila melanogaster as a model organism. Nerve-specific small GTPases were silenced and overexpressed. We examined the effects of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, we also studied the activation of small GTPases in a Parkinson's disease model. Our results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system. In the future, it might be interesting to assess the effect of Rab2 and Arl8 coactivation on autophagy, and it would also be worthwhile to validate these findings in a mammalian model and human cell lines. Molecules that specifically inhibit Rab2 or Arl8 serve as potent drug candidates to modulate the activity of the autophagic process in treating neurodegenerative pathologies. In the future, it would be reasonable to investigate which GAP enzyme can inhibit Rab2 or Arl8 specifically, but not affect Rab7, with similar medical purposes.

Plant UVRAG interacts with ATG14 to regulate autophagosome maturation and geminivirus infection.

Autophagy is an essential degradation pathway that assists eukaryote survival under multiple stress conditions. Autophagosomes engulfing cargoes accomplish degradation only when they have matured through fusing with lysosomes or vacuoles. However, the molecular machinery mediating autophagosome maturation in plants remains unknown. Using the combined approaches of mass spectrometry, biochemistry, reverse genetics and microscopy, we uncover that UVRAG, a subunit of the class III phosphatidylinositol 3-kinase complexes in Nicotiana benthamiana, plays an essential role in autophagsome maturation via ATG14-assisted recruitment to autophagosomes and by facilitating RAB7 activation. An interaction between N. benthamiana UVRAG and ATG14 was observed in vitro and in vivo, which strikingly differed from their mutually exclusive appearance in different PI3KC3 complexes in yeast and mammals. This interaction increased the localisation of UVRAG on autophagosomes and enabled the convergence of autophagic and late endosomal structures, where they contributed to fusions between these two types of organelles by recruiting the essential membrane fusion factors RAB7 GTPase and the homotypic fusion and protein sorting (HOPS) complex. In addition, we uncovered a joint contribution of ATG14 and UVRAG to geminiviral infection, beyond autophagy. Our study provides insights into the mechanisms of autophagosome maturation in plants and expands the understanding of organisations and roles of the PI3KC3 complexes.

Arabidopsis HOPS subunit VPS41 carries out plant-specific roles in vacuolar transport and vegetative growth.

The homotypic fusion and protein sorting (HOPS) complex is a conserved, multi-subunit tethering complex in eukaryotic cells. In yeast and mammalian cells, the HOPS subunit vacuolar protein sorting-associated protein 41 (VPS41) is recruited to late endosomes after Ras-related protein 7 (Rab7) activation and is essential for vacuole fusion. However, whether VPS41 plays conserved roles in plants is not clear. Here, we demonstrate that in the model plant Arabidopsis (Arabidopsis thaliana), VPS41 localizes to distinct condensates in root cells in addition to its reported localization at the tonoplast. The formation of condensates does not rely on the known upstream regulators but depends on VPS41 self-interaction and is essential for vegetative growth regulation. Genetic evidence indicates that VPS41 is required for both homotypic vacuole fusion and cargo sorting from the adaptor protein complex 3, Rab5, and Golgi-independent pathways but is dispensable for the Rab7 cargo inositol transporter 1. We also show that VPS41 has HOPS-independent functions in vacuolar transport. Taken together, our findings indicate that Arabidopsis VPS41 is a unique subunit of the HOPS complex that carries out plant-specific roles in both vacuolar transport and developmental regulation.

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a–VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome–lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.

HOPS-associated neurological disorders (HOPSANDs): linking endolysosomal dysfunction to the pathogenesis of dystonia.

The homotypic fusion and protein sorting (HOPS) complex is the structural bridge necessary for the fusion of late endosomes and autophagosomes with lysosomes. Recent publications linked mutations in genes encoding HOPS complex proteins with the aetiopathogenesis of inherited dystonias (i.e. VPS16, VPS41, and VPS11). Functional and microstructural studies conducted on patient-derived fibroblasts carrying mutations of HOPS complex subunits displayed clear abnormalities of the lysosomal and autophagic compartments. We propose to name this group of diseases HOPS-associated neurological disorders (HOPSANDs), which are mainly characterized by dystonic presentations. The delineation of HOPSANDs further confirms the connection of lysosomal and autophagic dysfunction with the pathogenesis of dystonia, prompting researchers to find innovative therapies targeting this pathway.

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1‐dependent TFEB/TFE3 regulation

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662*;VPS41c.1423‐2A>G and VPS41R662*) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41‐depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson’s disease, co‐expression of VPS41S285P/VPS41R662* abolished the neuroprotective function of VPS41 against α‐synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

A Novel Homozygous VPS11 Variant May Cause Generalized Dystonia.

In this work, we describe the association of a novel homozygous VPS11 variant with adult‐onset generalized dystonia, providing a detailed clinical report and biological evidence of disease mechanism. Vps11 is a subunit of the homotypic fusion and protein sorting (HOPS) complex, which promotes the fusion of late endosomes and autophagosomes with the lysosome. Functional studies on mutated fibroblasts showed marked lysosomal and autophagic abnormalities, which improved after overexpression of the wild type Vps11 protein. In conclusion, a deleterious VPS11 variant, damaging the autophagic and lysosomal pathways, is the probable genetic cause of a novel form of generalized dystonia. ANN NEUROL 2021;89:834–839

ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation.

Autophagy acts as a cellular surveillance mechanism to combat invading pathogens. Viruses have evolved various strategies to block autophagy and even subvert it for their replication and release. Here, we demonstrated that ORF3a of the COVID-19 virus SARS-CoV-2 inhibits autophagy activity by blocking fusion of autophagosomes/amphisomes with lysosomes. The late endosome-localized ORF3a directly interacts with and sequestrates the homotypic fusion and protein sorting (HOPS) component VPS39, thereby preventing HOPS complex from interacting with the autophagosomal SNARE protein STX17. This blocks assembly of the STX17-SNAP29-VAMP8 SNARE complex, which mediates autophagosome/amphisome fusion with lysosomes. Expression of ORF3a also damages lysosomes and impairs their function. SARS-CoV-2 virus infection blocks autophagy, resulting in accumulation of autophagosomes/amphisomes, and causes late endosomal sequestration of VPS39. Surprisingly, ORF3a from the SARS virus SARS-CoV fails to interact with HOPS or block autophagy. Our study reveals a mechanism by which SARS-CoV-2 evades lysosomal destruction and provides insights for developing new strategies to treat COVID-19.

Cargo Release from Myosin V Requires the Convergence of Parallel Pathways that Phosphorylate and Ubiquitylate the Cargo Adaptor

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.

Filoviruses Use the HOPS Complex and UVRAG To Traffic to Niemann-Pick C1 Compartments during Viral Entry.

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.

A trafficome-wide RNAi screen reveals deployment of early and late secretory host proteins and the entire late endo-/lysosomal vesicle fusion machinery by intracellular Salmonella.

The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation of SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies.

Disruption of vacuolar protein sorting components of the HOPS complex leads to enhanced secretion of recombinant proteins in Pichia pastoris

BackgroundThe yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases prevented this mis-sorting and resulted in higher levels of secreted heterologous protein. Inspired by the positive results, we now set out to investigate the influence of further parts of the vacuolar pathway, namely the Cvt-pathway and the homotypic fusion and protein sorting (HOPS) complex.ResultsStrains impaired in the Cvt pathway (∆atg11, ∆atg8) had no effect on secretion of the model protein carboxylesterase (CES), but resulted in lower secretion levels of the antibody fragment HyHEL-Fab. Disruption of genes involved in the HOPS complex led to vacuole-like compartments of the B category of vps mutants, which are characteristic for the deleted genes YPT7, VPS41 and VAM6. In particular ∆ypt7 and ∆vam6 strains showed an improvement in secreting the model proteins HyHEL-Fab and CES. Additional disruption of the vacuolar protease Pep4 and the potential protease Vps70 led to even further enhanced secretion in ∆ypt7 and ∆vam6 strains. Nevertheless, intracellular product accumulation was still observed. Therefore, the secretory route was strengthened by overexpression of early or late secretory genes in the vacuolar sorting mutants. Thereby, overexpression of Sbh1, a subunit of the ER translocation pore, significantly increased HyHEL-Fab secretion, leading up to fourfold higher extracellular Fab levels in the ∆ypt7 strain. The beneficial impact on protein secretion and the suitability of these strains for industrial applicability was confirmed in fed-batch cultivations.ConclusionsDisruption of genes involved in the HOPS complex, especially YPT7, has a great influence on the secretion of the two different model proteins HyHEL-Fab and CES. Therefore, disruption of HOPS genes shows a high potential to increase secretion of other recombinant proteins as well. Secretion of HyHEL-Fab was further enhanced when overexpressing secretion enhancing factors. As the positive effect was also present in fed-batch cultivations, these modifications likely have promising industrial relevance.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts—a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and—common to sphingolipid diseases—abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated β-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.

Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila

In the endocytic pathway of animals, two related complexes, called CORVET (class C core vacuole/endosome transport) and HOPS (homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease, including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes, but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent, while CORVET-specific subunits have proliferated. VPS8 (vacuolar protein sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup, including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, Δvps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst-sorting receptor Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating that target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.