ER–mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13

The endoplasmic reticulum–mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole–mitochondria and to vacuole–nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition–regulated organelle network.     

XK is a partner for VPS13A: a molecular link between Chorea-Acanthocytosis and McLeod Syndrome

Vps13 is a highly conserved lipid transfer protein found at multiple interorganelle membrane contact sites where it mediates distinct processes. In yeast, recruitment of Vps13 to different contact sites occurs via various partner proteins. In humans, four VPS13 family members, A–D, are associated with different diseases. In particular, vps13A mutants result in the neurodegenerative disorder Chorea-Acanthocytosis (ChAc). ChAc phenotypes resemble those of McLeod Syndrome, caused by mutations in the XK gene, suggesting that XK could be a partner protein for VPS13A. XK does, in fact, exhibit hallmarks of a VPS13A partner: it forms a complex with VPS13A in human cells and, when overexpressed, relocalizes VPS13A from lipid droplets to subdomains of the endoplasmic reticulum. Introduction of two different ChAc disease-linked missense mutations into VPS13A prevents this XK-induced relocalization. These results suggest that dysregulation of a VPS13A-XK complex is the common basis for ChAc and McLeod Syndrome.     

VPS13D bridges the ER to mitochondria and peroxisomes via Miro

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.     

Yeast and other lower eukaryotic organisms for studies of Vps13 proteins in health and disease

Human Vps13 proteins are associated with several diseases, including the neurodegenerative disorder Chorea‐acanthocytosis (ChAc), yet the biology of these proteins is still poorly understood. Studies in Saccharomyces cerevisiae, Dictyostelium discoideum, Tetrahymena thermophila and Drosophila melanogaster point to the involvement of Vps13 in cytoskeleton organization, vesicular trafficking, autophagy, phagocytosis, endocytosis, proteostasis, sporulation and mitochondrial functioning. Recent findings show that yeast Vps13 binds to phosphatidylinositol lipids via 4 different regions and functions at membrane contact sites, enlarging the list of Vps13 functions. This review describes the great potential of simple eukaryotes to decipher disease mechanisms in higher organisms and highlights novel insights into the pathological role of Vps13 towards ChAc.      

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.     

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.     

Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.     

Cnm1: A bridge between mitochondria and nuclear ER

Few membrane contact sites have been defined at the molecular level. By using a high-throughput, microscopy-based screen, Eisenberg-Bord, Zung et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202104100) identify Cnm1 as a novel tethering protein that mediates contact between mitochondria and the nuclear ER in response to phospholipid levels.

Organelles communicate through the exchange of biological materials by vesicular trafficking or at sites of close membrane apposition known as membrane contact sites (MCSs). While the molecular machinery mediating vesicular trafficking has been well characterized, our knowledge of the molecules involved in forming and regulating MCSs is limited. MCSs physically tether two or more organelles via protein–protein or protein–lipid interactions, contain defined proteomes, and perform specific biological functions (1). While MCSs have been appreciated microscopically since the 1950s, only recently have advances in technology permitted the discovery of the molecular composition of some MCSs (2). A major breakthrough occurred when a synthetic biology screen identified the ER–mitochondria encounter structure (ERMES), which forms an MCS between the ER and mitochondria (3). ERMES has since been shown to be involved in phospholipid transport between mitochondria and the ER (4). While ERMES is one of the best characterized MCSs, there are still many questions as to the precise molecules being transported at ER–mitochondria contacts and how directionality of transport is achieved. Subsequent studies using split fluorescent proteins revealed that nearly all organelles appear to form MCSs of some kind (5). Thus, despite progress in defining the components and functions of a few MCSs, there are still many MCSs whose molecular identities are completely unknown.Recently, a study in mammalian cells identified an MCS between the nucleus and mitochondria that plays a role in adapting cells to stress via the mitochondrial retrograde signaling response (6). The proteins that form this MCS are not conserved in yeast, however, suggesting that alternative mechanisms for nucleus–mitochondria contacts exist in other organisms. In this issue, Eisenberg-Bord, Zung et al., set out to identify proteins involved in forming an MCS between mitochondria and the nuclear ER that is distinct from ERMES-mediated ER–mitochondria contacts (7). First, high-resolution cryo-electron tomographs revealed that mitochondria form contacts with the nucleus that have an average separation of ∼20 nm, which is within the expected range for a bona fide MCS (1). To identify the molecular composition of this contact site, the authors generated a synthetic reporter that is specific to nucleus-mitochondria contacts by fusing one part of a split fluorescent protein to an outer mitochondrial membrane protein and the other to a peripheral nuclear protein. A high-throughput, microscopy-based genetic screen was then used to compare the localization of the synthetic reporter to fluorescently tagged versions of all yeast proteins. Candidates were refined by determining which proteins caused an expansion of the nucleus–mitochondria contact site upon overexpression, a phenotype that has been observed with other MCS proteins (8). Based on these results, the best candidate for a molecular tether between mitochondria and the nucleus was Ybr063c.Ybr063c is a 46-kD nonessential protein of uncharacterized function that contains predicted transmembrane domains. The authors first demonstrated that Ybr063c is an integral membrane protein residing on the nuclear membrane. In support of Ybr063c forming a nucleus–mitochondria contact site that is distinct from ERMES, Ybr063c did not colocalize with ERMES subunits nor did overexpression of Ybr063c alter the size of ERMES patches. Remarkably, overexpression of Ybr063c resulted in the mitochondrial network becoming tightly associated with the nuclear membrane. Based on these results, the authors concluded that Ybr063c functions as a molecular tether between mitochondria and the nucleus and the protein was renamed Cnm1 for contact nucleus mitochondria 1.Through further genetic screens, Eisenberg-Bord, Zung et al., identified several genes that are required to cluster mitochondria around the nucleus when Cnm1 is overexpressed. Interestingly, several of these genes are known to function in phosphatidylcholine (PC) metabolism. Deletion of these components resulted in a decrease in Cnm1 expression, which alters the extent of nucleus-mitochondria contacts. Overexpression of Cnm1 in genetic conditions that reduce PC levels resulted in exaggerated growth defects. These results raise the possibility that Cnm1-mediated nuclear–mitochondria contacts may be involved in the transport of PC from the ER to mitochondria. Thus, while the functional importance is unknown, Cnm1-mediated nuclear–mitochondria contacts respond to PC levels.The genetic screens also identified a single resident mitochondrial protein, Tom70, as affecting the ability of overexpressed Cnm1 to cluster mitochondria around the nucleus. Subsequent experiments demonstrated that localization of Cnm1 to the nuclear membrane and Tom70 to the mitochondrial membrane is required to tether mitochondria to the nucleus upon overexpression of Cnm1. Thus, Cnm1 and Tom70 mediate an MCS between mitochondria and the nucleus.The identification of Cnm1-mediated nucleus–mitochondria contacts opens many questions about the function and composition of the contact site and how it operates in the broader context of mitochondrial–nuclear communication. While identifying the functions of MCSs has proven challenging, the genetic screens conducted in this study provide an excellent starting point by elucidating a link between Cnm1 and PC metabolism. The authors propose that Cnm1-mediated contacts could function in the direct transport of PC from the ER to mitochondria (Fig. 1). In this model, ERMES, which likely functions in earlier steps of PC synthesis by transporting phosphatidylethanolamine (PE) or phosphatidylserine (PS), would have a distinct but related function in organizing and maintaining a pipeline for the transport of lipids between the ER and mitochondria (Fig. 1). This model is speculative, however, and future experiments will be necessary to define the role of Cnm1 in PC metabolism.Open in a separate windowFigure 1.The ER and vacuole form multiple MCSs with mitochondria in budding yeast. The ER is depicted in green, and the mitochondrial network is depicted in gray. ERMES mediates an MCS between tubular ER and mitochondria. In addition to functions that are distinct from lipid trafficking, ERMES-mediated MCSs likely function to transport PS or PE between the organelles. Cnm1 mediates an MCS specifically between the nuclear ER and mitochondria and potentially functions in PC transport. The Vps13-Mcp1 vCLAMP mediates an MCS between mitochondria and the vacuole that likely functions in lipid transport and may have redundant functions with ERMES. The Vps39-Tom40 vCLAMP is a separate MCS between mitochondria and the vacuole that responds to different stress conditions, though its function is unknown.There is a growing body of evidence that two organelles can form multiple MCSs that are spatially and functionally distinct. In addition to ERMES and Cnm1-mediated mitochondria–ER contacts, in yeast, two distinct MCSs have been described between mitochondria and the vacuole that are referred to as vacuolar and mitochondrial patches, or vCLAMPs. One, mediated by Vam6 and Tom40, has been implicated in responding to cellular stress while the other, mediated by Mcp1 and Vps13, may have overlapping functions with the ERMES complex (8, 9; Fig. 1). Interestingly, many of the proteins present at MCSs have been shown to be multifunctional (2). For example, the vCLAMP component Vam6 is also a subunit of the homotypic fusion and protein-sorting (HOPS) complex while its binding partner Tom40 is the central subunit of the translocase of outer membrane (TOM) complex (8). Thus, while these complexes have distinct biological functions in vacuolar protein sorting and mitochondrial protein import respectively, individual subunits have moonlighting functions in the formation, and perhaps function, of MCSs. Eisenberg-Bord, Zung et al., now reveal that Tom70, another component of the TOM complex, also plays a role in the formation of nucleus–mitochondria contacts. This raises the exciting possibility that cells use these multifunctional proteins to coordinate functions such as mitochondrial protein import with lipid trafficking. A crucial next step will be to determine how the multiple functions of these proteins are coordinated to maintain organelle homeostasis.Nuclear–mitochondrial communication is a critical aspect of eukaryotic cellular life that allows cells to adapt to different environmental conditions and energy needs. A breakdown in communication between mitochondria and the nucleus has been implicated in several diseases, including cancers (10). The formation of a nucleus–mitochondria MCS likely facilitates the exchange of lipids or small molecules that stimulate signaling pathways to help cells respond to environmental changes or mitochondrial damage (6, 7). Identifying the molecules that regulate these contacts and clarifying the physiological contexts under which these contacts function is crucial to our understanding of human disease. Thus, the identification of a nucleus-mitochondria MCS represents a significant breakthrough in our understanding of nucleus–mitochondria communication.     

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.     

A Conserved Function in Phosphatidylinositol Metabolism for Mammalian Vps13 Family Proteins

The Vps13 protein family is highly conserved in eukaryotic cells. In humans, mutations in the gene encoding the family member VPS13A lead to the neurodegenerative disorder chorea-acanthocytosis. In the yeast Saccharomyces cerevisiae, there is just a single version of VPS13, thereby simplifying the task of unraveling its molecular function(s). While VPS13 was originally identified in yeast by its role in vacuolar sorting, recent studies have revealed a completely different function for VPS13 in sporulation, where VPS13 regulates phosphatidylinositol-4-phosphate (PtdIns(4)P) levels in the prospore membrane. This discovery raises the possibility that the disease phenotype associated with vps13A mutants in humans is due to misregulation of PtdIns(4)P in membranes. To determine whether VPS13A affects PtdIns(4)P in membranes from mammalian neuronal cells, phosphatidylinositol phosphate pools were compared in PC12 tissue culture cells in the absence or presence of VPS13A. Consistent with the yeast results, the localization of PtdIns(4)P is specifically altered in VPS13A knockdown cells while other phosphatidylinositol phosphates appear unaffected. In addition, VPS13A is necessary to prevent the premature degeneration of neurites that develop in response to Nerve Growth Factor. The regulation of PtdIns(4)P is therefore a conserved function of the Vps13 family and may play a role in the maintenance of neuronal processes in mammals.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Biochemical Analyses of Human IST1 and Its Function in Cytokinesis

The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5–Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1–VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.     

VPS13D interacts with VCP/p97 and negatively regulates endoplasmic reticulum–mitochondria interactions

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and mitochondria are emerging as critical hubs for diverse cellular events, and alterations in the extent of these contacts are linked to neurodegenerative diseases. However, the mechanisms that control ER–mitochondria interactions are so far elusive. Here, we demonstrate a key role of vacuolar protein sorting–associated protein 13D (VPS13D) in the negative regulation of ER–mitochondria MCSs. VPS13D suppression results in extensive ER–mitochondria tethering, a phenotype that can be substantially rescued by suppression of the tethering proteins VAPB and PTPIP51. VPS13D interacts with valosin-containing protein (VCP/p97) to control the level of ER-resident VAPB at contacts. VPS13D is required for the stability of p97. Functionally, VPS13D suppression leads to severe defects in mitochondrial morphology, mitochondrial cellular distribution, and mitochondrial DNA synthesis. Together, our results suggest that VPS13D negatively regulates the ER–mitochondria MCSs, partially through its interactions with VCP/p97.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

Vps9 Family Protein Muk1 Is the Second Rab5 Guanosine Nucleotide Exchange Factor in Budding Yeast

VPS9 domains can act as guanosine nucleotide exchange factors (GEFs) against small G proteins of the Rab5 family. Saccharomyces cerevisiae vps9Δ mutants have trafficking defects considerably less severe than multiple deletions of the three cognate Rab5 paralogs (Vps21, Ypt52, and Ypt53). Here, we show that Muk1, which also contains a VPS9 domain, acts as a second GEF against Vps21, Ypt52, and Ypt53. Muk1 is partially redundant with Vps9 in vivo, with vps9Δ muk1Δ double mutant cells displaying hypersensitivity to temperature and ionic stress, as well as profound impairments in endocytic and Golgi endosome trafficking, including defects in sorting through the multivesicular body. Cells lacking both Vps9 and Muk1 closely phenocopy double and triple knock-out strains lacking Rab5 paralogs. Microscopy and overexpression experiments demonstrate that Vps9 and Muk1 have distinct localization determinants. These experiments establish Muk1 as the second Rab5 GEF in budding yeast.     

An Arabidopsis VPS45p Homolog Implicated in Protein Transport to the Vacuole

The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.     

Identification of a conserved motif required for Vps35p/Vps26p interaction and assembly of the retromer complex

The retromer complex is a conserved cytoplasmic coat complex that mediates the endosome-to-Golgi retrieval of vacuole/lysosome hydrolase receptors in yeast and mammals. The recognition of cargo proteins by the retromer is performed by the Vps35p/VPS35 (where Vps is vacuolar protein sorting) component, which together with Vps26p/VPS26 and Vps29p/VPS29, forms the cargo-selective subcomplex. In this report, we have identified a highly-conserved region of Vps35p/VPS35 that is essential for the interaction with Vps26p/VPS26 and for assembly of the retromer complex. Mutation of residues within the conserved region results in Vps35p/VPS35 mutants, which cannot bind to Vps26p/VPS26 and are not efficiently targeted to the endosomal membrane. These data implicate Vps26p/VPS26 in regulating Vps35p/VPS35 membrane association and therefore suggest a role for Vps26p/VPS26 in cargo recognition.     

Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans

Sec1/Munc‐18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps‐33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps‐33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS‐33.1 resulted in embryonic lethality. By contrast, vps‐33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm‐specific organelle. The endocytosis defect in the vps‐33.1 mutant was not restored by the expression of VPS‐33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS‐33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS‐33.2 has tissue/organelle specific functions in C. elegans.      

A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery.

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.     

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.