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 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.

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.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

A guanine nucleotide exchange factor (GEF) limits Rab GTPase-driven membrane fusion.

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF.

Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans.

blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.

Structure of the endosomal CORVET tethering complex.

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.

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.

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.

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.

Who's in control? Principles of Rab GTPase activation in endolysosomal membrane trafficking and beyond.

The eukaryotic endomembrane system consists of multiple interconnected organelles. Rab GTPases are organelle-specific markers that give identity to these membranes by recruiting transport and trafficking proteins. During transport processes or along organelle maturation, one Rab is replaced by another, a process termed Rab cascade, which requires at its center a Rab-specific guanine nucleotide exchange factor (GEF). The endolysosomal system serves here as a prime example for a Rab cascade. Along with endosomal maturation, the endosomal Rab5 recruits and activates the Rab7-specific GEF Mon1-Ccz1, resulting in Rab7 activation on endosomes and subsequent fusion of endosomes with lysosomes. In this review, we focus on the current idea of Mon1-Ccz1 recruitment and activation in the endolysosomal and autophagic pathway. We compare identified principles to other GTPase cascades on endomembranes, highlight the importance of regulation, and evaluate in this context the strength and relevance of recent developments in in vitro analyses to understand the underlying foundation of organelle biogenesis and maturation.

Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer.

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a 'closed' and an 'open' conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution.

Proteins are part of the building blocks of life and are essential for structure, function and regulation of every cell, tissue and organ of the body. Proteins adopt different conformations to work efficiently within the various environments of a cell. They can also switch between shapes. One way to monitor how proteins change their shapes involves energy transfer. This approach can measure how close two proteins, or two parts of the same protein, are, by using dye labels that respond to each other when they are close together. For example, in a method called FRET, one dye label absorbs light and transfers the energy to the other label, which emits it as a different color of light. However, FRET only works over short distances (less than 10nm apart or 1/100,000th of a millimeter), so it is not useful for larger proteins. Here, Füllbrunn, Li et al. developed a method called GIET that uses graphene to analyze the dynamic structures of proteins on membrane surfaces. Graphene is a type of carbon nanomaterial that can absorb energy from dye labels and could provide a way to study protein interactions over longer distances. Graphene was deposited on a glass surface where it was coated with single layer of membrane, which could then be used to capture specific proteins. The results showed that GIET worked over longer distances (up to 30 nm) than FRET and could be used to study proteins attached to the membrane around graphene. Füllbrunn, Li et al. used it to examine a specific complex of proteins called HOPS, which is linked to multiple diseases, including Ebola, measuring distances between the head or tail of HOPS and the membrane to understand protein shapes. This revealed that HOPS adopts an upright position on membranes and alternates between open and closed shapes. The study of Füllbrunn, Li et al. highlights the ability of GIET to address unanswered questions about the function of protein complexes on membrane surfaces and sheds new light on the structural dynamics of HOPS in living cells. As it allows protein interactions to be studied over much greater distances, GIET could be a powerful new tool for cell biology research. Moreover, graphene is also useful in electron microscopy and both approaches combined could achieve a detailed structural picture of proteins in action.

A conserved and regulated mechanism drives endosomal Rab transition.

Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway.

Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: effect of repositioning within helix 4 of subunit a and helix 2 of subunit c.

FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((alphabeta)3gammadeltaepsilon) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10gammaepsilon relative to (alphabeta)3deltaab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but "horizontal" repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of "vertical" repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.

Rab GTPase Function in Endosome and Lysosome Biogenesis.

Eukaryotic cells maintain a highly organized endolysosomal system. This system regulates the protein and lipid content of the plasma membrane, it participates in the intracellular quality control machinery and is needed for the efficient removal of damaged organelles. This complex network comprises an endosomal membrane system that feeds into the lysosomes, yet also allows recycling of membrane proteins, and probably lipids. Moreover, lysosomal degradation provides the cell with macromolecules for further growth. In this review, we focus primarily on the role of the small Rab GTPases Rab5 and Rab7 as organelle markers and interactors of multiple effectors on endosomes and lysosomes and highlight their role in membrane dynamics, particularly fusion along the endolysosomal pathway.

Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure.

During autophagy, a newly formed double membrane surrounds its cargo to generate the so-called autophagosome, which then fuses with a lysosome after closure. Previous work implicated that endosomal Rab7/Ypt7 associates to autophagosomes prior to their fusion with lysosomes. Here, we unravel how the Mon1-Ccz1 guanosine exchange factor (GEF) acting upstream of Ypt7 is specifically recruited to the pre-autophagosomal structure under starvation conditions. We find that Mon1-Ccz1 directly binds to Atg8, the yeast homolog of the members of the mammalian LC3 protein family. This requires at least one LIR motif in the Ccz1 C-terminus, which is essential for autophagy but not for endosomal transport. In agreement, only wild-type, but not LIR-mutated Mon1-Ccz1 promotes Atg8-dependent activation of Ypt7. Our data reveal how GEF targeting can specify the fate of a newly formed organelle and provide new insights into the regulation of autophagosome-lysosome fusion.

Multivalent Rab interactions determine tether-mediated membrane fusion.

Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes.