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.

C9orf72-catalyzed GTP loading of Rab39A enables HOPS-mediated membrane tethering and fusion in mammalian autophagy.

The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a "hook-up" model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation.

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and the vacuole.

The Saccharomyces cerevisiae casein kinase protein Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates of this protein. We found that endogenously tagged Yck3 localized not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localized to both endosomes and vacuoles, and Yck3 controlled this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, whereas its non-phosphorylatable SA variant strongly localized to endosomes, similar to what was observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying the Rab7-like protein Ypt7. Modeling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which were both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.

Structure of the metazoan Rab7 GEF complex Mon1-Ccz1-Bulli.

The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1-Ccz1-Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy-Inturned-Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy-Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation.

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

The nutrient-responsive CDK Pho85 primes the Sch9 kinase for its activation by TORC1.

Yeast cells maintain an intricate network of nutrient signaling pathways enabling them to integrate information on the availability of different nutrients and adjust their metabolism and growth accordingly. Cells that are no longer capable of integrating this information, or that are unable to make the necessary adaptations, will cease growth and eventually die. Here, we studied the molecular basis underlying the synthetic lethality caused by loss of the protein kinase Sch9, a key player in amino acid signaling and proximal effector of the conserved growth-regulatory TORC1 complex, when combined with either loss of the cyclin-dependent kinase (CDK) Pho85 or loss of its inhibitor Pho81, which both have pivotal roles in phosphate sensing and cell cycle regulation. We demonstrate that it is specifically the CDK-cyclin pair Pho85-Pho80 or the partially redundant CDK-cyclin pairs Pho85-Pcl6/Pcl7 that become essential for growth when Sch9 is absent. Interestingly, the respective three CDK-cyclin pairs regulate the activity and distribution of the phosphatidylinositol-3 phosphate 5-kinase Fab1 on endosomes and vacuoles, where it generates phosphatidylinositol-3,5 bisphosphate that serves to recruit both TORC1 and its substrate Sch9. In addition, Pho85-Pho80 directly phosphorylates Sch9 at Ser726, and to a lesser extent at Thr723, thereby priming Sch9 for its subsequent phosphorylation and activation by TORC1. The TORC1-Sch9 signaling branch therefore integrates Pho85-mediated information at different levels. In this context, we also discovered that loss of the transcription factor Pho4 rescued the synthetic lethality caused by loss of Pho85 and Sch9, indicating that both signaling pathways also converge on Pho4, which appears to be wired to a feedback loop involving the high-affinity phosphate transporter Pho84 that fine-tunes Sch9-mediated responses.

Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to distinct organelles by a synergistic protein and lipid code.

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization, but the mechanisms that underly MC1 localization have not been fully elucidated. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids, and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. Membrane binding enhances MC1 activity predominantly by increasing enzyme and substrate concentration on the membrane, but interaction with recruiter proteins can further stimulate the guanine nucleotide exchange factor. Our data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. In conclusion, we reveal the molecular basis for how MC1 is adapted to recognize distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model for how the complex is regulated and activated independently in different functional contexts.

Molecular insights into endolysosomal microcompartment formation and maintenance.

The endolysosomal system of eukaryotic cells has a key role in the homeostasis of the plasma membrane, in signaling and nutrient uptake, and is abused by viruses and pathogens for entry. Endocytosis of plasma membrane proteins results in vesicles, which fuse with the early endosome. If destined for lysosomal degradation, these proteins are packaged into intraluminal vesicles, converting an early endosome to a late endosome, which finally fuses with the lysosome. Each of these organelles has a unique membrane surface composition, which can form segmented membrane microcompartments by membrane contact sites or fission proteins. Furthermore, these organelles are in continuous exchange due to fission and fusion events. The underlying machinery, which maintains organelle identity along the pathway, is regulated by signaling processes. Here, we will focus on the Rab5 and Rab7 GTPases of early and late endosomes. As molecular switches, Rabs depend on activating guanine nucleotide exchange factors (GEFs). Over the last years, we characterized the Rab7 GEF, the Mon1-Ccz1 (MC1) complex, and key Rab7 effectors, the HOPS complex and retromer. Structural and functional analyses of these complexes lead to a molecular understanding of their function in the context of organelle biogenesis.

--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9's role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery.

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery.

Our cells break down the nutrients that they receive from the body to create the building blocks needed to keep us alive. This is done by compartments called lysosomes that are filled with a cocktail of proteins called enzymes, which speed up the breakdown process. Lysosomes are surrounded by a membrane, a barrier of fatty molecules that protects the rest of the cell from being digested. When new nutrients reach the cell, they travel to the lysosome packaged in vesicles, which have their own fatty membrane. To allow the nutrients to enter the lysosome without creating a leak, the membranes of the vesicles and the lysosome must fuse. The mechanism through which these membranes fuse is not fully clear. It is known that both fusing membranes must contain proteins called SNAREs, which wind around each other when they interact. However, this alone is not enough. Other proteins are also required to tether the membranes together before they fuse. To understand how these tethers play a role, Shvarev, Schoppe, König et al. studied the structure of the HOPS complex from yeast. This assembly of six proteins is vital for lysosomal fusion and, has a composition similar to the equivalent complex in humans. Using cryo-electron microscopy, a technique that relies on freezing purified proteins to image them with an electron microscope and reveal their structure, allowed Shvarev, Schoppe, König et al. to provide a model for how HOPS interacts with SNAREs and membranes. In addition to HOPS acting as a tether to bring the membranes together, it can also bind directly to SNAREs. This creates a bridge that allows the proteins to wrap around each other, driving the membranes to fuse. HOPS is a crucial component in the cellular machinery, and mutations in the complex can cause devastating neurological defects. The complex is also targeted by viruses ­ such as SARS-CoV-2 ­ that manipulate HOPS to reduce its activity. Shvarev, Schoppe, König et al.'s findings could help researchers to develop drugs to maintain or recover the activity of HOPS. However, this will require additional information about its structure and how the complex acts in the biological environment of the cell.

Vesicle transport: Exocyst follows PIP2 to tether membranes.

A new study uses reconstituted, functional octameric exocyst complex to provide new insights into the assembly of this tethering complex and reveal how the activity of the lipid kinase PIP5K1C stimulated by Arf6 on exocytic vesicles allows for exocyst-mediated tethering at the plasma membrane.

The HOPS tethering complex is required to maintain signaling endosome identity and TORC1 activity.

The endomembrane system of eukaryotic cells is essential for cellular homeostasis during growth and proliferation. Previous work showed that a central regulator of growth, namely the target of rapamycin complex 1 (TORC1), binds both membranes of vacuoles and signaling endosomes (SEs) that are distinct from multivesicular bodies (MVBs). Interestingly, the endosomal TORC1, which binds membranes in part via the EGO complex, critically defines vacuole integrity. Here, we demonstrate that SEs form at a branch point of the biosynthetic and endocytic pathways toward the vacuole and depend on MVB biogenesis. Importantly, function of the HOPS tethering complex is essential to maintain the identity of SEs and proper endosomal and vacuolar TORC1 activities. In HOPS mutants, the EGO complex redistributed to the Golgi, which resulted in a partial mislocalization of TORC1. Our study uncovers that SE function requires a functional HOPS complex and MVBs, suggesting a tight link between trafficking and signaling along the endolysosomal pathway.

The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.

Lysosomes mediate degradation of macromolecules to their precursors for cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein and an effector of the minor Rab5 isoform Ypt52. Strikingly, bph1Δ mutant cells have lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy-inducing conditions. In agreement with this, proteomic analysis of bph1Δ vacuoles reveals an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1Δ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When Bph1 is lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways.

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.

Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes.

Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole.

The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.