Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Structure of membrane tethers and their role in fusion

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so‐called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.     

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion

Mitochondria are double‐membrane‐bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N‐terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane‐bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C‐terminal amphipathic tail of the Atlastin protein.     

Molecular basis for sterol transport by StART‐like lipid transfer domains

Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non‐vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a‐c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo‐bound and sterol‐bound forms, at 2.0 Å resolution. It folds into a truncated version of the steroidogenic acute regulatory protein‐related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3‐hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.     

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.     

Membrane scission driven by the PROPPIN Atg18

Sorting, transport, and autophagic degradation of proteins in endosomes and lysosomes, as well as the division of these organelles, depend on scission of membrane‐bound tubulo‐vesicular carriers. How scission occurs is poorly understood, but family proteins bind these membranes. Here, we show that the yeast PROPPIN Atg18 carries membrane scission activity. Purified Atg18 drives tubulation and scission of giant unilamellar vesicles. Upon membrane contact, Atg18 folds its unstructured CD loop into an amphipathic α‐helix that inserts into the bilayer. This allows the protein to engage its two lipid binding sites for PI3P and PI(3,5)P₂. PI(3,5)P₂ induces Atg18 oligomerization, which should concentrate lipid‐inserted α‐helices in the outer membrane leaflet and drive membrane tubulation and scission. The scission activity of Atg18 is compatible with its known roles in endo‐lysosomal protein trafficking, autophagosome biogenesis, and vacuole fission. Key features required for membrane tubulation and scission by Atg18 are shared by other PROPPINs, suggesting that membrane scission may be a generic function of this protein family.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Insights into the role of the membranes in Rab GTPase regulation

Rab GTPases are molecular switches with essential roles in mediating vesicular trafficking and establishing organelle identity. The conversion from the inactive, cytosolic to the membrane-bound, active species and back is tightly controlled by regulatory proteins. Recently, the roles of membrane properties and lipid composition of different target organelles in determining the activity state of Rabs have come to light. The investigation of several Rab guanine nucleotide exchange factors (GEFs) has revealed principles of how the recruitment via lipid interactions and the spatial confinement on the membrane surface contribute to spatiotemporal specificity in the Rab GTPase network. This paints an intricate picture of the control mechanisms in Rab activation and highlights the importance of the membrane lipid code in the organization of the endomembrane system.     

NbRABG3f,a member of Rab GTPase,is involved in Bamboo mosaic virus infection in Nicotiana benthamiana

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up‐regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up‐regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus‐induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane‐targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP‐NbRABG3f/T22N clearly co‐localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane‐targeting ability are crucial for BaMV accumulation at the cell level.     

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.     

Peroxisomes: membrane events accompanying peroxisome proliferation

Peroxisomes are ubiquitous organelles surrounded by a single membrane that display a variety of metabolic functions. These vary with the organism in which they occur and with environmental conditions. Peroxisomes multiply by division of existing organelles and can be formed from ER. The peroxisomal membrane, akin to the organelle itself, is a very dynamic structure that obtains building blocks from the ER. It can form diverse organized structures - lipid domains - that can be involved in regulation of various vesicle fusion processes. Additionally, this membrane may undergo extensive changes in lipid composition. We recently showed that upon proliferation the peroxisomal membrane changes its curvature in response to the activity of the peroxisomal membrane protein Pex11. Tubulation of the organelle may be important for efficient recruitment of GTPases from the dynamin protein family that is involved in organelle fission.     

Rafting through traffic: Membrane domains in cellular logistics

The intricate and tightly regulated organization of eukaryotic cells into spatially and functionally distinct membrane-bound compartments is a defining feature of complex organisms. These compartments are defined by their lipid and protein compositions, with their limiting membrane as the functional interface to the rest of the cell. Thus, proper segregation of membrane proteins and lipids is necessary for the maintenance of organelle identity, and this segregation must be maintained despite extensive, rapid membrane exchange between compartments. Sorting processes of high efficiency and fidelity are required to avoid potentially deleterious mis-targeting and maintain cellular function. Although much molecular machinery associated with membrane traffic (i.e. membrane budding/fusion/fission) has been characterized both structurally and biochemically, the mechanistic details underlying the tightly regulated distribution of membranes between subcellular locations remain to be elucidated. This review presents evidence for the role of ordered lateral membrane domains known as lipid rafts in both biosynthetic sorting in the late secretory pathway, as well as endocytosis and recycling to/from the plasma membrane. Although such evidence is extensive and the involvement of membrane domains in sorting is definitive, specific mechanistic details for raft-dependent sorting processes remain elusive.     

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.     

Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging from global carbon fixation to enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid‐based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best‐studied MCPs highlighting atomic‐level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications of an unusual compartment designed by nature to optimize metabolic processes within a cellular context.     

Changing phosphoinositides “on the fly”: how trafficking vesicles avoid an identity crisis

Joining an antagonistic phosphoinositide (PtdInsP) kinase and phosphatase into a single protein complex may regulate rapid and local PtdInsP changes. This may be important for processes such as membrane fission that require a specific PtdInsP and that are innately local and rapid. Such a complex could couple vesicle formation, with erasing of the identity of the donor organelle from the vesicle prior to its fusion with target organelles, thus preventing organelle identity intermixing. Coordinating signals are postulated to switch the relative activities of the kinase and phosphatase in a spatio‐temporal manner that matches membrane fission events. The discovery of two such complexes supports this hypothesis. One regulates the interconversion of phosphatidylinositol and PtdIns(3)P by joining the Vps34 PtdIns 3‐kinase and the myotubularin 3‐phosphatases. The other regulates the interconversion between PtdIns(3)P and PtdIns(3,5)P₂ through the Fab1/PIKfyve kinase and the Fig4/mFig4 phosphatase. These lipids are essential components of the endosomal identity code.     

Reconstitution of vesicular transport to Rab11-positive recycling endosomes in vitro

Rab GTPases are key regulators of vesicular protein transport in both the endocytic and exocytic pathways. In endocytosis and recycling, Rab11 plays a role in receptor recycling to plasma membrane via the pericentriolar recycling compartment. However, little is known about the molecular requirements and partners that promote transport through Rab11-positive recycling endosomes. Here, we report a novel approach to reconstitute transport to immunoabsorbed recycling endosomes in vitro. We show that transport is temperature-, energy-, and time-dependent and requires the presence of Rab proteins, as it is inhibited by the Rab-interacting protein Rab GDP-dissociation inhibitor that removes Rab proteins from the membrane. Cytochalasin D, a drug that blocks actin polymerization, inhibits the in vitro assay, suggesting that transport to recycling endosomes depends on an intact actin cytoskeleton. Using an affinity chromatography approach we show the identification of Rab11-interacting proteins including actin that stimulate transport to recycling endosomes in vitro.     

Rab proteins, connecting transport and vesicle fusion

Small GTPases of the Rab family control timing of vesicle fusion. Fusion of two vesicles can only occur when they have been brought into close contact. Transport by microtubule- or actin-based motor proteins will facilitate this process in vivo. Ideally, transport and vesicle fusion are linked activities. Active, GTP-bound Rab proteins dock on specific compartments and are therefore perfect candidates to control transport of the different compartments. Recently, a number of Rab proteins were identified that control motor protein recruitment to their specific target membranes. By cycling through inactive and active states, Rab proteins are able to control motor protein-mediated transport and subsequent fusion of intracellular structures in both spatial and timed manners.