The GTPase activating protein Gyp7 regulates Rab7/Ypt7 activity on late endosomes.

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.

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

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.

Cellular metabolism regulates contact sites between vacuoles and mitochondria.

Emerging evidence suggests that contact sites between different organelles form central hubs in the coordination of cellular physiology. Although recent work has emphasized the crucial role of the endoplasmic reticulum in interorganellar crosstalk, the cooperative behavior of other organelles is largely unexplored. Here, we identify a contact site named vCLAMP (vacuole and mitochondria patch) that integrates mitochondria with the lysosome-like vacuole and thus the endocytic pathway. vCLAMPs depend on the vacuolar HOPS tethering complex subunit Vps39/Vam6 and the Rab GTPase Ypt7, which also participate in membrane fusion at the vacuole. Intriguingly, vCLAMPs are located proximal to the ER-mitochondria encounter structure (ERMES) complexes, and an increase in vCLAMPs can rescue the growth defect of ERMES mutants. Importantly, the persistence of vCLAMPs is regulated by phosphorylation of Vps39 and is strongly reduced during respiratory growth. The identification of this organelle contact site reveals a physical and metabolic interconnection between the endocytic pathway and mitochondria.

Nanogold labeling of the yeast endosomal system for ultrastructural analyses.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.

Functional separation of endosomal fusion factors and the class C core vacuole/endosome tethering (CORVET) complex in endosome biogenesis.

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.

Suppression of α-synuclein toxicity and vesicle trafficking defects by phosphorylation at S129 in yeast depends on genetic context.

The aggregation of α-synuclein (αSyn) is a neuropathologic hallmark of Parkinson's disease and other synucleinopathies. In Lewy bodies, αSyn is extensively phosphorylated, predominantly at serine 129 (S129). Recent studies in yeast have shown that, at toxic levels, αSyn disrupts Rab homeostasis, causing an initial endoplasmic reticulum-to-Golgi block that precedes a generalized trafficking collapse. However, whether αSyn phosphorylation modulates trafficking defects has not been evaluated. Here, we show that constitutive expression of αSyn in yeast impairs late-exocytic, early-endocytic and/or recycling trafficking. Although members of the casein kinase I (CKI) family phosphorylate αSyn at S129, they attenuate αSyn toxicity and trafficking defects by an S129 phosphorylation-independent mechanism. Surprisingly, phosphorylation of S129 modulates αSyn toxicity and trafficking defects in a manner strictly determined by genetic background. Abnormal endosome morphology, increased levels of the endosome marker Rab5 and co-localization of mammalian CKI with αSyn aggregates are observed in brain sections from αSyn-overexpressing mice and human synucleinopathies. Our results contribute to evidence that suggests αSyn-induced defects in endocytosis, exocytosis and/or recycling of vesicles involved in these cellular processes might contribute to the pathogenesis of synucleinopathies.

The mitochondrial contact site complex, a determinant of mitochondrial architecture.

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high-resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA.

SNARE proteins are required for macroautophagy.

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

Adaptation of the Tokuyasu method for the ultrastructural study and immunogold labelling of filamentous fungi.

The study of filamentous fungi is fundamental not only to extend their biotechnological applications, but also to develop new drugs to fight pathological species. Morphological analyses are particularly relevant when investigating their development and differentiation. The need to maintain the orientation of hypahe and the presence of a cell wall, which hampers the sample infiltration with cryoprotectants and other reagents necessary to preserve the cell ultrastructure, creates difficulties with the use of electron microscopy (EM). Here, we present an immunoelectron microscopy (IEM) procedure that combines the Tokuyasu protocol adapted to yeast and the flat-embedding technique. While the first method leads to a fine resolution of the ultrastructure of Aspergillus nidulans because of both the cell wall permeabilization and the negative membrane coloration, the second permits us to preserve the spatial distribution of the hypahe of this fungus. The presented data demonstrate the advantages of this combination and the unprecedented potential of this relatively simple and rapid protocol in resolving the morphology of filamentous fungi and performing localization studies.

Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.

Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab guanosine triphosphatases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with the target membranes. Little is known about how tethering factors discriminate between different trafficking pathways, which may converge at the same organelle. In this paper, we describe a phosphorylation-based switch mechanism, which allows the homotypic vacuole fusion protein sorting effector subunit Vps41 to operate in two distinct fusion events, namely endosome-vacuole and AP-3 vesicle-vacuole fusion. Vps41 contains an amphipathic lipid-packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is inserted into the lipid bilayer and masks the binding motif for the δ subunit of the AP-3 complex, Apl5, without affecting the Vps41 function in endosome-vacuole fusion. At the much less curved vacuole, the ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the Apl5-binding site is exposed and allows AP-3 vesicles to bind to Vps41, followed by specific fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phosphorylation.

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

Exit from the Golgi is required for the expansion of the autophagosomal phagophore in yeast Saccharomyces cerevisiae.

The delivery of proteins and organelles to the vacuole by autophagy involves membrane rearrangements that result in the formation of large vesicles called autophagosomes. The mechanism underlying autophagosome biogenesis and the origin of the membranes composing these vesicles remains largely unclear. We have investigated the role of the Golgi complex in autophagy and have determined that in yeast, activation of ADP-ribosylation factor (Arf)1 and Arf2 GTPases by Sec7, Gea1, and Gea2 is essential for this catabolic process. The two main events catalyzed by these components, the biogenesis of COPI- and clathrin-coated vesicles, do not play a critical role in autophagy. Analysis of the sec7 strain under starvation conditions revealed that the autophagy machinery is correctly assembled and the precursor membrane cisterna of autophagosomes, the phagophore, is normally formed. However, the expansion of the phagophore into an autophagosome is severely impaired. Our data show that the Golgi complex plays a crucial role in supplying the lipid bilayers necessary for the biogenesis of double-membrane vesicles possibly through a new class of transport carriers or a new mechanism.

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.

Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure.

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system.

Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.

Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with high-pressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.

In vivo role of lipid adducts on Wingless.

Two lipids (palmitate and palmitoleic acid) are appended onto Wnt proteins. It has been suggested that palmitate is required for signalling, whereas palmitoleic acid is necessary for progression through the secretory pathway. By mutating the relevant amino acids, we have investigated how these adducts contribute to the secretion and signalling activity of Wingless, the main Drosophila member of the Wnt family. Analysis of Wingless with a Cysteine 93 to Alanine mutation ([C93A]Wingless) shows that palmitoylation is essential for signalling activity in vivo (as well as in cultured cells). Moreover, without palmitate, Wingless fails to reach the surface of imaginal disc cells and, as electron microscopy (EM) analysis suggests, appears to accumulate in the endoplasmic reticulum (ER). Artificial targeting of palmitate-deficient Wingless to the plasma membrane does not rescue signalling activity. Therefore, palmitate at C93 has a dual role: in secretion and signalling. From our analysis of [S239A]Wingless, which lacks a conserved residue shown to be acylated in Wnt3a, we infer that palmitoleic acid is not, as previously suggested, absolutely required for secretion. Nevertheless, this mutant has poor signalling activity, suggesting that palmitoleic acid contributes significantly to signalling. We suggest that the overall level of lipidation affects signalling activity.

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex.

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors.

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.