Nucleus-vacuole Junction Research Articles

The yeast VAPs Scs2 and Scs22 are required for NVJ integrity and micronucleophagy

Microautophagy degrades cargos in the vacuole by direct engulfment of the vacuolar membrane. Micronucleophagy selectively degrades a portion of the nucleus in budding yeast. The vacuole contacts the nucleus via the nucleus–vacuole junction (NVJ), and in micronucleophagy a portion of the nucleus containing nucleolar proteins is made to protrude into the vacuole at the NVJ, followed by abscission and degradation. Microautophagy and micronucleophagy are induced by inactivation of target of rapamycin complex 1 (TORC1) protein kinase after nutrient starvation. Here, we show that the VAMP-associated proteins (VAPs) Scs2 and its paralog Scs22 are required for NVJ integrity and micronucleophagic degradation of nucleolar proteins. On the other hand, nucleolar dynamics prerequisite for micronucleophagy were not impaired in VAP mutant cells. Finally, yeast VAPs were critical for viability during prolonged nutrient starvation. This study sheds light on the emerging role of VAP in adaptation in responses to nutrient starvation.

Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)–plasma membrane (PM) and ER–Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus–vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Membrane contact sites regulate vacuolar fission via sphingolipid metabolism.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Roles for inter‐organelle contacts in organizing metabolism

When faced with nutrient shortage, cells adapt by remodeling their metabolic pathways and organelles. I will discuss our work using budding yeast to dissect how nutrient shortage impacts the spatial organization of metabolism and lipid droplets. We find that in response to glucose restriction, yeast remodel their mevalonate pathway by spatially compartmentalizing its rate‐limiting enzyme, HMG‐CoA Reductase (HMGCR). HMGCR spatial compartmentalization occurs at a unique inter‐organelle contact site called the nucleus‐vacuole junction (NVJ). This spatial partitioning enhances HMGCR activity, driving mevalonate synthesis to enable cellular adaptation. Our work suggests a new use for an inter‐organelle contact site in the fine‐tuning of mevalonate metabolism during nutrient stress.Remarkably, we also find that glucose restriction drives the phase transition of lipids within lipid droplets (LDs), causing them to convert from a disordered to liquid‐crystalline phase. Mechanistically, we find that these liquid crystalline lattices (LCLs) within LDs require triglyceride lipolysis. We also find that LCL‐LDs exhibit changes to the LD surface proteome. Several known LD proteins redistribute from LDs to the ER network, whereas others remain on LCL‐LDs, suggesting phase transitions of LD lipids influences LD protein targeting. Global proteomics also reveals that triglycerides harvested from LDs fuel cellular energetics at peroxisomes and mitochondria. This indicates that glucose starvation induces inter‐organelle lipid flux while promoting lipid phase transitions within LDs.

Unexplored Cdc42 functions at the budding yeast nucleus suggested by subcellular localization

ABSTRACT In budding yeast, the Rho-family GTPase Cdc42 has several functions that depend on its subcellular localization and the cell cycle stage. During bud formation, Cdc42 localizes to the plasma membrane at the bud tip and bud neck where it carries out functions in actin polymerization, spindle positioning, and exocytosis to ensure proper polarity development. Recent live-cell imaging analysis revealed a novel localization of Cdc42 to a discrete intracellular focus associated with the vacuole and nuclear envelope. The discovery of this novel Cdc42 localization led to the identification of a new function in ESCRT-mediated nuclear envelope sealing. However, other aspects of this intracellular localization and its functional implications were not explored. Here, we further characterize the Cdc42 focus and present several novel observations that suggest possible additional Cdc42 functions at the nucleus, including nucleus-vacuole junction formation, nuclear envelope tethering, nuclear migration, and nucleopodia formation.

Partitioning of the mevalonate synthesizing enzymes at ER‐lysosome contacts influences their catalytic efficiency

The last decade of molecular biology is hallmarked by an extraordinary increase in our understanding of previously unappreciated spatial scales in biology. Enzymes and metabolites are partitioned into distinct membrane-bound organelles to spatially regulate metabolism and signaling. More recent discoveries have uncovered that enzymes can also inducibly or permanently compartmentalize within sub-organelle domains such as inter-organelle contact sites (1). However, a key knowledge gap is understanding role that enzyme partitioning plays on regulating enzyme function or pathway flux. Here, we performed a GFP-based screen in Saccharomyces cerevisiae to identify metabolic enzymes that may enrich at sub-organelle domains. We found that during Acute Glucose Restriction (AGR) the rate-limiting enzymes of sterol biogenesis, Hmg1 and Hmg2, partition at the Nucleus-Vacuole Junction (NVJ), an inter-organelle contact site between the nuclear envelope and yeast vacuole. NVJ partitioning of Hmg1/2 correlates with increased de-novo synthesis of sterol-esters. Additionally, Hmg1/2 partitioning can be blocked by deletion of the NVJ tether protein Nvj1. Remarkably, Hmg1/2 is less catalytically efficient in Nvj1 knock-out cells, suggesting a potential link between its spatial compartmentalization and increased enzymatic activity. We further demonstrate that Nvj1-mediated partitioning of Hmg1/2 during AGR contributes to cellular fitness, and coincides with ability to resume growth following glucose starvation. Overall, we have uncovered a previously underappreciated level of spatial regulation for Hmg1/2, and also identified a novel role for inter-organelle contact sites in the regulation of mevalonate metabolism.

Organelle homeostasis principles: How organelle quality control and inter-organelle crosstalk promote cell survival.

To survive, cells sense their surroundings and adapt to enable homeostasis. Studies dissecting this process reveal organizational principles, including quality-control pathways, changes to organelle shape, and inter-organelle communication, that facilitate metabolic or developmental remodeling. In this issue, several reviews discuss these organelle homeostasis principles and how they are altered in disease.

Sorting nexin Mdm1/SNX14 regulates nucleolar dynamics at the NVJ after TORC1 inactivation

The degradation of nucleolar proteins – nucleophagy – is elicited by nutrient starvation or the inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Prior to nucleophagy, nucleolar proteins migrate to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA (rRNA gene) repeat regions are condensed and escape towards NVJ-distal sites. This suggests that the NVJ controls nucleolar dynamics from outside of the nucleus after TORC1 inactivation, but its molecular mechanism is unclear. Here, we show that sorting nexin (SNX) Mdm1, an inter-organelle tethering protein at the NVJ, mediates TORC1 inactivation-induced nucleolar dynamics. Furthermore, Mdm1 was required for proper nucleophagic degradation of nucleolar proteins after TORC1 inactivation, where it was dispensable for the induction of nucleophagic flux itself. This indicated that nucleophagy and nucleolar dynamics are independently regulated by TORC1 inactivation. Finally, Mdm1 was critical for survival during nutrient starvation conditions. Mutations of SNX14, a human Mdm1 homolog, cause neurodevelopmental disorders. This study provides a novel insight into relationship between sorting nexin-mediated microautophagy and neurodevelopmental disorders.

The vacuole controls nucleolar dynamics and micronucleophagy via the NVJ

Chromosomes have their own territories and dynamically translocate in response to internal and external cues. However, whether and how territories and the relocation of chromosomes are controlled by other intracellular organelles remains unknown. Upon nutrient starvation and target of rapamycin complex 1 (TORC1) inactivation, micronucleophagy, which preferentially degrades nucleolar proteins, occurs at the nucleus–vacuole junction (NVJ) in budding yeast. Ribosomal DNA (rDNA) is condensed and relocated against the NVJ, whereas nucleolar proteins move towards the NVJ for micronucleophagic degradation, causing dissociation of nucleolar proteins from rDNA. These findings imply that the NVJ is the critical platform in the directional movements of rDNA and nucleolar proteins. Here, we show that cells lacking the NVJ (NVJΔ cells) largely lost rDNA condensation and rDNA–nucleolar protein separation after TORC1 inactivation. The macronucleophagy receptor Atg39, an outer nuclear membrane protein, accumulated at the NVJ and was degraded by micronucleophagy. These suggested that macronucleophagy is also dependent on the presence of the NVJ. However, micronucleophagy, but not macronucleophagy, was abolished in NVJΔ cells. This study clearly demonstrated that vacuoles controls intranuclear events, nucleolar dynamics, from outside of the nucleus via the NVJ under the control of TORC1.

Cdc14 protein phosphatase and topoisomerase II mediate rDNA dynamics and nucleophagic degradation of nucleolar proteins after TORC1 inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.

Nutrient Signaling, Stress Response, and Inter-organelle Communication Are Non-canonical Determinants of Cell Fate

Isogenic cells manifest distinct cellular fates for a single stress; however, the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergoes distinct fates, becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, senescence occurs even in the absence of lipid droplets. Importantly, we identify the nutrient-sensing kinase Rim15 as a key biomarker predicting cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contains determinants influencing post-stress fate and demonstrate that specific nutrient signaling, stress response, trafficking, and inter-organelle biomarkers are early indicators for long-term fate outcomes.

Nutrient Signaling, Stress Response, and Interorganelle Communication are Non-Canonical Determinants of Cell Fate

Isogenic cells can manifest distinct cellular fates for a single stress, however the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergo distinct fates becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, we also show senescence occurs in the absence of lipid droplets. Importantly, we identify the nutrient-sensing linked kinase Rim15 as a key biomarker that predicts cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contain determinants that influence their post-stress fate, and demonstrate that specific nutrient signaling, stress-response, endomembrane trafficking, and inter-organelle tether biomarkers are early indicators for long-term fate outcomes.

Nucleophagy-Implications for Microautophagy and Health.

Nucleophagy, the selective subtype of autophagy that targets nuclear material for autophagic degradation, was not only shown to be a model system for the study of selective macroautophagy, but also for elucidating the role of the core autophagic machinery within microautophagy. Nucleophagy also emerged as a system associated with a variety of disease conditions including cancer, neurodegeneration and ageing. Nucleophagic processes are part of natural cell development, but also act as a response to various stress conditions. Upon releasing small portions of nuclear material, micronuclei, the autophagic machinery transfers these micronuclei to the vacuole for subsequent degradation. Despite sharing many cargos and requiring the core autophagic machinery, recent investigations revealed the aspects that set macro- and micronucleophagy apart. Central to the discrepancies found between macro- and micronucleophagy is the nucleus vacuole junction, a large membrane contact site formed between nucleus and vacuole. Exclusion of nuclear pore complexes from the junction and its exclusive degradation by micronucleophagy reveal compositional differences in cargo. Regarding their shared reliance on the core autophagic machinery, micronucleophagy does not involve normal autophagosome biogenesis observed for macronucleophagy, but instead maintains a unique role in overall microautophagy, with the autophagic machinery accumulating at the neck of budding vesicles.

Atg21 organizes Atg8 lipidation at the contact of the vacuole with the phagophore

ABSTRACT Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12–Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation. Abbreviations: FCCS: fluorescence cross correlation spectroscopy; NVJ: nucleus-vacuole junction; PAS: phagophore assembly site; PE: phosphatidylethanolamine;PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; VICS: vacuole isolation membrane contact site.

Mechanistic dissection of macro- and micronucleophagy

ABSTRACT Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes. Abbreviations: DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.

Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

ABSTRACT Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19–33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways. Abbreviations Ape1: aminopeptidase I; ARM: armadillo repeat; Atg: autophagy-related; AUC: analytical ultracentrifugation; Cvt: cytoplasm-to-vacuole targeting; DIC: differential interference contrast; GFP: green fluorescent protein; GST: glutathione-S-transferase; ITC: isothermal titration calorimetry; NVJ: nucleus-vacuole junction; PDB: protein data bank; PMN: piecemeal microautophagy of the nucleus; prApe1: precursor Ape1; RMSD: root-mean-square deviation; SAXS: small-angle X-ray scattering; SD-N: nitrogen starvation medium; SEC: size-exclusion chromatography; tAtg13: Atg13 construct comprising residues 567–695; tNvj1: Nvj1 construct comprising residues 229–321; tVac8: Vac8 construct comprising residues 10–515; Vac8: vacuole related 8

RDNA Condensation Promotes rDNA Separation from Nucleolar Proteins Degraded for Nucleophagy after TORC1 Inactivation

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase induce nucleophagy preferentially degrading only nucleolar components in budding yeast. Nucleolar proteins are relocated to sites proximal to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA, which is embedded in the nucleolus under normal conditions, moves to NVJ-distal regions, causing rDNA dissociation from nucleolar proteins after TORC1 inactivation. This repositioning ismediated via chromosome linkage INM protein (CLIP)-cohibin complexes that tether rDNA to the inner nuclear membrane. Here, we show that TORC1 inactivation-induced rDNA condensation promotes the repositioning of rDNA and nucleolar proteins. Defects in condensin, Rpd3-Sin3 histone deacetylase (HDAC), and high-mobility group protein 1 (Hmo1), which are involved in TORC1 inactivation-induced rDNA condensation, compromised the repositioning and nucleophagic degradation of nucleolar proteins, although rDNA still escaped from nucleophagic degradation in these mutants. We propose a model in which rDNA condensation after TORC1 inactivation generates a motive force for the repositioning of rDNA and nucleolar proteins.

A reversible liquid drop aggregation controls glucose response in yeast.

Glucose is the preferred carbon of the yeast Saccharomyces cerevisiae. Depletion of glucose activates SNF1 (yeast AMP-activated protein kinase-AMPK), allowing cells to switch from fermentation to respiration. We have recently characterized the mechanism by which SNF1 activity is regulated by the Std1 protein, and its regulator Sip5. The hitherto uncharacterized protein kinase Vhs1 phosphorylates Sip5 in response to glucose availability, disengaging it from Std1 and promoting the sequestering of the SNF1 activator out of the nucleus into cytoplasmic puncta. These aggregates, which have the properties of liquid drops, and not of amyloids, reside in the nucleus-vacuole junction. The process is reversible, and Std1 puncta dissolve when glucose becomes scarce again. This reversible process requires protein chaperones, similar to the aggregation of toxic or misfolded proteins such as those associated with Huntington's Chorea, Alzheimer's and CJD diseases. Our results thus reveal a regulated, non-pathological, physiological role of protein aggregation that controls a major metabolic cellular pathway.

An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.