Henning Arlt

The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome

Organelles of the endomembrane system need to counterbalance fission and fusion events to maintain their surface-to-volume ratio. At the late mammalian endosome, the Rab GTPase Rab7 is a major regulator of fusion, whereas the homologous yeast protein Ypt7 seems to be restricted to the vacuole surface. Here, we present evidence that Ypt7 is recruited to and acts on late endosomes, where it affects multiple trafficking reactions. We show that overexpression of Ypt7 results in expansion and massive invagination of the vacuolar membrane, which requires cycling of Ypt7 between GDP- and GTP-bound states. Invaginations are blocked by ESCRT, CORVET and retromer mutants, but not by autophagy or AP-3 mutants. We also show that Ypt7–GTP specifically binds to the retromer cargo-recognition subcomplex, which – like its cargo Vps10 – is found on the vacuole upon Ypt7 overproduction. Our data suggest that Ypt7 functions at the late endosome to coordinate retromer-mediated recycling with the fusion of late endosomes with vacuoles.

The CORVET Subunit Vps8 Cooperates with the Rab5 Homolog Vps21 to Induce Clustering of Late Endosomal Compartments

Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.

Functional Separation of Endosomal Fusion Factors and the Class C Core Vacuole/Endosome Tethering (CORVET) Complex in Endosome Biogenesis

Background: The interdependence of the endosomal fusion factors CORVET and Vac1 has not been addressed. Results: CORVET can be separated from other endocytic fusion factors on the basis of ultrastructure, localization, and trafficking. Conclusion: CORVET acts independently of the Vac1 tether and requires activated Rab5 homologs for localization. Significance: Our data reveal a unique role of CORVET in sorting of biosynthetic cargo to the vacuole. Transport along the endolysosomal system requires multiple fusion events at early and late endosomes. Deletion of several endosomal fusion factors, including the Vac1 tether and the Class C core vacuole/endosome tethering (CORVET) complex-specific subunits Vps3 and Vps8, results in a class D vps phenotype. As these mutants have an apparently similar defect in endosomal transport, we asked whether CORVET and Vac1 could still act in distinct tethering reactions. Our data reveal that CORVET mutants can be rescued by Vac1 overexpression in the endocytic pathway but not in CPY or Cps1 sorting to the vacuole. Moreover, when we compared the ultrastructure, CORVET mutants were most similar to deletions of the Rab Vps21 and its guanine nucleotide exchange factor Vps9 and different from vac1 deletion, indicating separate functions. Likewise, CORVET still localized to endosomes even in the absence of Vac1, whereas Vac1 localization became diffuse in CORVET mutants. Importantly, CORVET localization requires the Rab5 homologs Vps21 and Ypt52, whereas Vac1 localization is strictly Vps21-dependent. In this context, we also uncover that Muk1 can compensate for loss of Vps9 in CORVET localization, indicating that two Rab5 guanine nucleotide exchange factors operate in the endocytic pathway. Overall, our study reveals a unique role of CORVET in the sorting of biosynthetic cargo to the vacuole/lysosome.

The I-BAR protein Ivy1 is an effector of the Rab7 GTPase Ypt7 involved in vacuole membrane homeostasis

ABSTRACT Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show that Ivy1, the yeast homolog of mammalian missing-in-metastasis (MIM), is a vacuolar effector of Ypt7-GTP and interacts with the EGO/ragulator complex, an activator of the target of rapamycin kinase complex 1 (TORC1) on vacuoles. Loss of Ivy1 does not affect EGO vacuolar localization and function. In combination with the deletion of individual subunits of the V-ATPase, however, we observed reduced TORC1 activity and massive enlargement of the vacuole surface. Consistent with this, Ivy1 localizes to invaginations at the vacuole surface and on liposomes in a phosphoinositide- and Ypt7-GTP-controlled manner, which suggests a role in microautophagy. Our data, thus, reveal that Ivy1 is a novel regulator of vacuole membrane homeostasis with connections to TORC1 signaling. Summary: The I-BAR protein Ivy1 is a new effector of the yeast GTPase Ypt7, which localizes to membrane intrusions and affects the homeostasis of vacuole membranes.

An overexpression screen in Saccharomyces cerevisiae identifies novel genes that affect endocytic protein trafficking.

A large number of proteins involved in the biogenesis of yeast endosomes and vacuoles have been identified based on screens that scored for inactivation of proteins. Such screens may, however, miss important regulators of the pathway. Here, we present a visual screen in which we examined the effects on vacuole morphology if any of the 6153 yeast open reading frames was overexpressed. Using a progressive screening procedure, we could identify a total of 53 genes. Among the most striking endosomal proteins are the CORVET/HOPS subunits Vps3, Vps18 and Vps39 and the putative tethering inhibitor Ivy1. Furthermore, six endosomal sorting complex related to transport (ESCRT) proteins led to altered vacuole morphology if overproduced. Among the novel proteins, we identify Yer128w as an endosomal protein that interacts with the AAA‐ATPase Vps4, and therefore named it Vfa1 (Vps Four‐Associated 1). We present evidence on the possible role of these novel proteins in trafficking to the vacuole. Our data provide novel insights into the regulation of protein trafficking.

Retromer and the dynamin Vps1 cooperate in the retrieval of transmembrane proteins from vacuoles

ABSTRACT Endosomes are dynamic organelles that need to combine the ability to successfully deliver proteins and lipids to the lysosome-like vacuole, and recycle others to the Golgi or the plasma membrane. We now show that retromer, which is implicated in retrieval of proteins from endosomes to the Golgi or to the plasma membrane, can act on vacuoles. We explore its function using an assay that allows us to dissect the required cofactors during recycling. We demonstrate that recycling of the transmembrane receptor Vps10 from vacuoles requires the retromer, the dynamin-like Vps1, and the Rab7 GTPase Ypt7. Retromer and Vps1 leave the vacuole together with the cargo, whereas Ypt7 stays behind, in agreement with its regulatory function. Recycled cargo then accumulates at endosomes and later at the Golgi, implying consecutive sorting steps to the final destination. Our data further suggest that retromer and Vps1 are essential to maintain vacuole membrane organization. Taken together, our data demonstrate that retromer can cooperate with Vps1 and the Rab Ypt7 to clear the vacuole of selected membrane proteins.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

Endosomal sorting requires consecutive steps of membrane remodeling and fusion in the course of endosomal maturation. Tracing of cargo relative to machinery reveals similar temporal localization of ESCRT and endosomal fusion machinery, which precedes the retromer complex. However, blocking fusion with the vacuole does not impair maturation.

Retromer-driven membrane tubulation separates endosomal recycling from Rab7/Ypt7-dependent fusion

How does a Rab function in both recycling and fusion? An endosomal subcomplex of the SNX-BAR retromer can bind to Ypt7 and compete with the HOPS complex. Assembly of the full retromer then results in displacement of Ypt7. These data explain how domain formation and Ypt7 participation can be coordinated.

Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling

The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate ‘membrane compartment occupied by Wsc1’ (MCW) shows moderate overlap with other Wsc‐type sensors, but not with those of the Mid‐type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine‐rich head group near the N‐terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis.

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.