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.

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

VPS41-mediated incomplete autophagy aggravates cadmium-induced apoptosis in mouse hepatocytes.

Exposure to cadmium (Cd), an environmental heavy metal contaminant, is a serious threat to global health that increases the burden of liver diseases. Autophagy and apoptosis are important in Cd-induced liver injury. However, the regulatory mechanisms involved in the progression of Cd-induced liver damage are poorly understood. Herein, we investigated the role of vacuolar protein sorting 41 (VPS41) in Cd-induced autophagy and apoptosis in hepatocytes. We used targeted VPS41 regulation to elucidate the mechanism of Cd-induced hepatotoxicity. Our data showed that Cd triggered incomplete autophagy by downregulating VPS41, aggravating Cd-induced hepatocyte apoptosis. Mechanistically, Cd-induced VPS41 downregulation interfered with the mTORC1-TFEB/TFE3 axis, leading to an imbalance in autophagy initiation and termination and abnormal activation of autophagy. Moreover, Cd-induced downregulation of VPS41 inhibited autophagosome-lysosome fusion, leading to blocked autophagic flux. This triggers incomplete autophagy, which causes excessive P62 accumulation, accelerating Caspase-9 (CASP9) cleavage. Incomplete autophagy blocks clearance of cleaved CASP9 (CL-CASP9) via the autophagic pathway, promoting apoptosis. Notably, VPS41 overexpression alleviated Cd-induced incomplete autophagy and apoptosis, independent of the homotypic fusion and protein sorting complex. This study provides a new mechanistic understanding of the relationship between autophagy and apoptosis, suggesting that VPS41 is a new therapeutic target for Cd-induced liver damage.

Targeting VPS41 induces methuosis and inhibits autophagy in cancer cells.

The homotypic fusion and vacuole protein sorting (HOPS) complex mediates membrane trafficking involved in endocytosis, autophagy, lysosome biogenesis, and phagocytosis. Defects in HOPS subunits are associated with various forms of cancer, but their potential as drug targets has rarely been examined. Here, we identified vacuolar protein sorting-associated protein 41 homolog (VPS41), a subunit of the HOPS complex, as a target of methyl 2,4-dihydroxy-3-(3-methyl-2-butenyl)-6-phenethylbenzoate (DMBP), a natural small molecule with preferable anticancer activity. DMBP induced methuosis and inhibited autophagic flux in cancer cells by inhibiting the function of VPS41, leading to the restrained fusion of late endosomes and autophagosomes with lysosomes. Moreover, DMBP effectively inhibited metastasis in a mouse metastatic melanoma model. Collectively, the current work revealed that targeting VPS41 would provide a valuable method of inhibiting cancer proliferation through methuosis.

Circ-Vps41 positively modulates Syp and its overexpression improves memory ability in aging mice.

Introduction: Age is an established risk factor for neurodegenerative disorders. Aging-related cognitive decline is a common cause of memory impairment in aging individuals, in which hippocampal synaptic plasticity and hippocampus-dependent memory formation are damaged. Circular RNAs (circRNAs) have been reported in many cognitive disorders, but their role in aging-related memory impairment is unclear.Methods: In this study, we aimed to investigate the effects of circ-Vps41 on aging-related hippocampus-dependent memory impairment and explore the potential mechanisms. Here, D-galactose was used to produce a conventional aging model resulting in memory dysfunction. Results: Circ-Vps41 was significantly downregulated in D-galactose-induced aging in vitro and in vivo. The overexpression of circ-Vps41 could upregulate synaptophysin (Syp), thereby promoting the synaptic plasticity and alleviating cognitive impairment in aging mice. Mechanistically, we found that circ-Vps41 upregulated Syp expression by physically binding to miR-24-3p. Moreover, the miR-24-3p mimics reversed the circ-Vps41 overexpression-induced increase in Syp expression. Discussion: Overexpression of circ-Vps41 alleviated the synaptic plasticity and memory dysfunction via the miR-24-3p/Syp axis. These findings revealed circ-Vps41 regulatory network and provided new insights into its potential mechanisms for improving aging-related learning and memory impairment.

Nrf2 improves hippocampal synaptic plasticity, learning and memory through the circ-Vps41/miR-26a-5p/CaMKIV regulatory network.

Antioxidant response transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2/Nfe2l2) is a neuroprotective agent in learning and memory impairment. This study provides a new perspective to explore the regulatory mechanisms of Nrf2. Here, we found that Nrf2 regulated circular RNA circ-Vps41 to increase hippocampal synaptic plasticity; Nrf2 bound the Vps41 promoter to activate transcription of the Vps41 gene and promote expression of circ-Vps41; circ-Vps41 positively correlated with Nrf2, synaptic plasticity, and learning and memory but negatively correlated with reactive oxygen species; and Nrf2 promoted CaMKIV expression by increasing levels of circ-Vps41, which can absorb miR-26a-5p that targets CaMKIV. Our findings revealed a new circRNA-based regulatory network regulated by Nrf2 and provided novel insights into the potential mechanism involved in the improvement of learning and memory impairment.

TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei.

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket ("big eye" phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

Bi-allelic variants in HOPS complex subunit VPS41 cause cerebellar ataxia and abnormal membrane trafficking.

Membrane trafficking is a complex, essential process in eukaryotic cells responsible for protein transport and processing. Deficiencies in vacuolar protein sorting (VPS) proteins, key regulators of trafficking, cause abnormal intracellular segregation of macromolecules and organelles and are linked to human disease. VPS proteins function as part of complexes such as the homotypic fusion and vacuole protein sorting (HOPS) tethering complex, composed of VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41. The HOPS-specific subunit VPS41 has been reported to promote viability of dopaminergic neurons in Parkinson's disease but to date has not been linked to human disease. Here, we describe five unrelated families with nine affected individuals, all carrying homozygous variants in VPS41 that we show impact protein function. All affected individuals presented with a progressive neurodevelopmental disorder consisting of cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus. Zebrafish disease modelling supports the involvement of VPS41 dysfunction in the disorder, indicating lysosomal dysregulation throughout the brain and providing support for cerebellar and microglial abnormalities when vps41 was mutated. This provides the first example of human disease linked to the HOPS-specific subunit VPS41 and suggests the importance of HOPS complex activity for cerebellar function.

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.

AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering.

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.

CmVPS41 Is a General Gatekeeper for Resistance to Cucumber Mosaic Virus Phloem Entry in Melon.

Melon production is often compromised by viral diseases, which cannot be treated with chemicals. Therefore, the use of genetic resistances is the main strategy for generating crops resistant to viruses. Resistance to Cucumber mosaic virus (CMV) in melon is scarcely described in few accessions. Until recently, the only known resistant accessions were Freeman's Cucumber and PI 161375, cultivar Songwhan Charmi (SC). Resistance to CMV in melon is recessive and generally oligogenic and quantitative. However, in SC, the resistance to CMV strains of subgroup II is monogenic, depending only on one gene, cmv1, which is able to stop CMV movement by restricting the virus to the bundle sheath cells and preventing a systemic infection. This restriction depends on the viral movement protein (MP). Chimeric viruses carrying the MP of subgroup II strains, like the strain LS (CMV-LS), are restricted in the bundle sheath cells, whereas those carrying MP from subgroup I, like the strain FNY (CMV-FNY), are able to overcome this restriction. cmv1 encodes a vacuolar protein sorting 41 (CmVPS41), a protein involved in the transport of cargo proteins from the Golgi to the vacuole through late endosomes. We have analyzed the variability of the gene CmVPS41 in a set of 52 melon accessions belonging to 15 melon groups, both from the spp melo and the spp agrestis. We have identified 16 different haplotypes, encoding 12 different CmVPS41 protein variants. Challenging members of all haplotypes with CMV-LS, we have identified nine new resistant accessions. The resistance correlates with the presence of two mutations, either L348R, previously found in the accession SC and present in other three melon genotypes, or G85E, present in Freeman's Cucumber and found also in four additional melon genotypes. Moreover, the new resistant accessions belong to three different melon horticultural groups, Conomon, Makuwa, and Dudaim. In the new resistant accessions, the virus was able to replicate and move cell to cell, but was not able to reach the phloem. Therefore, resistance to phloem entry seems to be a general strategy in melon controlled by CmVPS41. Finally, the newly reported resistant accessions broaden the possibilities for the use of genetic resistances in new melon breeding strategies.

Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation.

Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.

Requirement of the isocitrate lyase gene ICL1 for VPS41-mediated starvation response in Cryptococcus neoformans.

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.

AtVPS41-mediated endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis.

In flowering plants, extensive male-female interactions are required for successful fertilization in which various signaling cascades are involved. Prevacuolar compartments (PVC) and vacuoles are two types of subcellular compartments that terminate signal transduction by sequestrating signaling molecules in yeast and mammalian cells; however, the manner in which they might be involved in male-female interactions in plants is unknown. In this study, we identified Arabidopsis thaliana vacuolar protein sorting 41 (AtVPS41), encoded by a single-copy gene with sequence similarity to yeast Vps41p, as a new factor controlling pollen tube-stigma interaction. Loss of AtVPS41 function disrupted penetration of pollen tubes into the transmitting tissue and thus led to failed male transmission. In the pollen tubes, AtVPS41 protein is associated with PVCs and the tonoplast. We demonstrate that AtVPS41 is required for the late stage of the endocytic pathway (i.e., endomembrane trafficking from PVCs to vacuoles) because internalization of cell-surface molecules was normal in the vps41-deficient pollen tubes, whereas PVC-to-vacuole trafficking was impaired. We further show that the CHCR domain is required for subcellular localization and biological functioning of AtVPS41. These results indicate that the AtVPS41-mediated late stage of the endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis.

Genes involved in the WNT and vesicular trafficking pathways are associated with melanoma predisposition.

Multifactorial predisposition to melanoma includes genes involved in pigmentation, immunity and DNA repair. Nonetheless, missing heritability in melanoma is still important. We studied the role of 335 candidate SNPs in melanoma susceptibility by using a dedicated chip and investigating 110 genes involved in different pathways. A discovery set was comprised of 1069 melanoma patients and 925 controls from France. Data were replicated using validation phases II (1085 cases and 801 controls from Spain) and III (1808 cases and 1894 controls from Germany and a second set of Spanish samples). In addition, an exome sequencing study was performed in three high-risk French melanoma families. Nineteen SNPs in 17 genes were initially associated with melanoma in the French population. Six SNPs were replicated in phase II, including two new SNPs in the WNT3 (rs199524) and VPS41 (rs11773094) genes. The role of VPS41 and WNT3 was confirmed in a meta-analysis (3940 melanoma cases and 3620 controls) with two-side p values of 0.002, (OR = 0.86) and 4.07 × 10(-10) (OR = 0.80), respectively. Exome sequencing revealed a non-synonymous VPS41 variant in one family that was shown to be strongly associated with familial melanoma (OR = 4.46, p = 0.001) in an independent sample of 178 melanoma families. WNT3 belongs to WNT pathway known to play a crucial role in melanoma, whereas VPS41 regulates vesicular trafficking and is thought to play a role in pigmentation. Our work identified two new pathways involved in melanoma predisposition. These results may be useful in the future for identifying individuals highly predisposed to melanoma.

Tracking of the dynamic localization of the Rab-specific HOPS subunits reveal their distinct interaction with Ypt7 and vacuoles.

Endosomal and vacuole fusion depends on the two homologous tethering complexes CORVET and HOPS. HOPS binds the activated Rab GTPase Ypt7 via two distinct subunits, Vps39 and Vps41. To understand the participation and possible polarity of Vps41 and Vps39 during tethering, we used an in vivo approach. For this, we established the ligand-induced relocalization to the plasma membrane, using the Mon1-Ccz1 GEF complex that activates Ypt7 on endosomes. We then employed slight overexpression to compare the mobility of the HOPS-specific Vps41 and Vps39 subunits during this process. Our data indicate an asymmetry in the Rab-specific interaction of the two HOPS subunits: Vps39 is more tightly bound to the vacuole, and relocalizes the entire vacuole to the plasma membrane, whereas Vps41 behaved like the more mobile subunit. This is due to their specific Rab binding, as the mobility of both subunits was similar in ypt7∆ cells. In contrast, both HOPS subunits were far less mobile if tagged endogenously, suggesting that the entire HOPS complex is tightly bound to the vacuole in vivo. Similar results were obtained for the endosomal association of CORVET, when we followed its Rab-specific subunit Vps8. Our data provide in vivo evidence for distinct Rab specificity within HOPS, which may explain its function during tethering, and indicate that these tethering complexes are less mobile within the cell than previously anticipated.

Vacuoles in mammals: a subcellular structure indispensable for early embryogenesis.

A vacuole is a membrane-bound subcellular structure involved in intracellular digestion. Instead of the large "vacuolar" organelles that are found in plants and fungi, animal cells possess lysosomes that are smaller in size and are enriched with hydrolytic enzymes similar to those found in the vacuoles. Large vacuolar structures are often observed in highly differentiated mammalian tissues such as embryonic visceral endoderm and absorbing epithelium. Vacuoles/lysosomes share a conserved mechanism of biogenesis, and they are at the terminal of the endocytic pathways, Recent genetic studies of the mammalian orthologs of Vam/Vps genes, which have essential functions for vacuole assembly, revealed that the dynamics of vacuoles/lysosomes are important for tissue differentiation and patterning through regulation of various molecular signaling events in mammals.