RESUMO
Endosomes are central protein-sorting stations at the crossroads of numerous membrane trafficking pathways in all eukaryotes. They have a key role in protein homeostasis and cellular signalling and are involved in the pathogenesis of numerous diseases. Endosome-associated protein assemblies or coats collect transmembrane cargo proteins and concentrate them into retrieval domains. These domains can extend into tubular carriers, which then pinch off from the endosomal membrane and deliver the cargoes to appropriate subcellular compartments. Here we discuss novel insights into the structure of a number of tubular membrane coats that mediate the recruitment of cargoes into these carriers, focusing on sorting nexin-based coats such as Retromer, Commander and ESCPE-1. We summarize current and emerging views of how selective tubular endosomal carriers form and detach from endosomes by fission, highlighting structural aspects, conceptual challenges and open questions.
RESUMO
Proteins exit from endosomes through tubular carriers coated by retromer, a complex that impacts cellular signaling, lysosomal biogenesis and numerous diseases. The coat must overcome membrane tension to form tubules. We explored the dynamics and driving force of this process by reconstituting coat formation with yeast retromer and the BAR-domain sorting nexins Vps5 and Vps17 on oriented synthetic lipid tubules. This coat oligomerizes bidirectionally, forming a static tubular structure that does not exchange subunits. High concentrations of sorting nexins alone constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, oligomers of retromer must bind and interconnect the sorting nexins to drive constriction. Constricting less curved membranes into tubes, which requires more energy, coincides with an increased surface density of retromer on the sorting nexin layer. Retromer-mediated crosslinking of sorting nexins at variable densities may thus tune the energy that the coat can generate to deform the membrane. In line with this, genetic ablation of retromer oligomerization impairs endosomal protein exit in yeast and human cells.
Assuntos
Saccharomyces cerevisiae , Nexinas de Classificação , Humanos , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Constrição , Endossomos/metabolismoRESUMO
Endo-lysosomal compartments exchange proteins by fusing, fissioning, and through endosomal transport carriers. Thereby, they sort many plasma membrane receptors and transporters and control cellular signaling and metabolism. How the membrane fission events are catalyzed is poorly understood. Here, we identify the novel CROP complex as a factor acting at this step. CROP joins members of two protein families: the peripheral subunits of retromer, a coat forming endosomal transport carriers, and membrane inserting PROPPINs. Integration into CROP potentiates the membrane fission activity of the PROPPIN Atg18 on synthetic liposomes and confers strong preference for binding PI(3,5)P2 , a phosphoinositide required for membrane fission activity. Disrupting CROP blocks fragmentation of lysosome-like yeast vacuoles in vivo. CROP-deficient mammalian endosomes accumulate micrometer-long tubules and fail to export cargo, suggesting that carriers attempt to form but cannot separate from these organelles. PROPPINs compete for retromer binding with the SNX-BAR proteins, which recruit retromer to the membrane during the formation of endosomal carriers. Transition from retromer-SNX-BAR complexes to retromer-PROPPIN complexes might hence switch retromer activities from cargo capture to membrane fission.
Assuntos
Endossomos , Nexinas de Classificação , Animais , Endossomos/metabolismo , Lisossomos/metabolismo , Mamíferos , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Nexinas de Classificação/metabolismoRESUMO
PROPPINs constitute a conserved protein family with multiple members being expressed in many eukaryotes. PROPPINs have mainly been investigated for their role in autophagy, where they co-operate with several core factors for autophagosome formation. Recently, novel functions of these proteins on endo-lysosomal compartments have emerged. PROPPINs support the division of these organelles and the formation of tubulo-vesicular cargo carriers that mediate protein exit from them, such as those generated by the Retromer coat. In both cases, PROPPINs provide membrane fission activity. Integrating information from yeast and human cells this review summarizes the most important molecular features that allow these proteins to facilitate membrane fission and thus provide a critical element to endo-lysosomal protein traffic.
Assuntos
Autofagia , Endossomos , Lisossomos , Humanos , Lisossomos/metabolismo , Autofagia/fisiologia , Endossomos/metabolismo , Animais , Transporte Proteico , Membranas Intracelulares/metabolismoRESUMO
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.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas de Fluorescência Verde/metabolismo , Lipídeos/química , Proteínas de Membrana/química , Mutação/genética , Peptídeos/química , Fosfatos de Fosfatidilinositol/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Cloreto de Sódio/farmacologia , Imagem com Lapso de Tempo , Lipossomas Unilamelares/metabolismo , Vacúolos/metabolismoRESUMO
Phagocytic cells take up, kill and digest microbes by a process called phagocytosis. To this end, these cells bind the particle, rearrange their actin cytoskeleton, and orchestrate transport of digestive factors to the particle-containing phagosome. The mammalian lysosomal membrane protein LIMP-2 (also known as SCARB2) and CD36, members of the class B of scavenger receptors, play a crucial role in lysosomal enzyme trafficking and uptake of mycobacteria, respectively, and generally in host cell defences against intracellular pathogens. Here, we show that the Dictyostelium discoideum LIMP-2 homologue LmpA regulates phagocytosis and phagolysosome biogenesis. The lmpA knockdown mutant is highly affected in actin-dependent processes, such as particle uptake, cellular spreading and motility. Additionally, the cells are severely impaired in phagosomal acidification and proteolysis, likely explaining the higher susceptibility to infection with the pathogenic bacterium Mycobacterium marinum, a close cousin of the human pathogen Mycobacterium tuberculosis Furthermore, we bring evidence that LmpB is a functional homologue of CD36 and specifically mediates uptake of mycobacteria. Altogether, these data indicate a role for LmpA and LmpB, ancestors of the family of which LIMP-2 and CD36 are members, in lysosome biogenesis and host cell defence.
Assuntos
Dictyostelium/fisiologia , Proteínas de Membrana Lisossomal/metabolismo , Mycobacterium marinum/fisiologia , Fagocitose , Proteínas de Protozoários/metabolismo , Receptores de Lipoproteínas/metabolismo , Antígenos CD36/genética , Dictyostelium/genética , Dictyostelium/microbiologia , Humanos , Proteínas de Membrana Lisossomal/genética , Proteínas de Protozoários/genética , Receptores de Lipoproteínas/genética , Receptores Depuradores/genéticaRESUMO
Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components.
Assuntos
Membrana Celular/metabolismo , Dictyostelium/metabolismo , Fagocitose , Fagossomos/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Integrinas/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Ligação Proteica , Transporte Proteico , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Proteins that contain Eps15 homology domains (EHDs) in their C-terminus are newly identified key regulators of endosomal membrane trafficking. Here, we show that D. discoideum contains a single EHD protein (referred to as EHD) that localizes to endosomal compartments and newly formed phagosomes. We provide the first evidence that EHD regulates phagosome maturation. Deletion of EHD results in defects in intraphagosomal proteolysis and acidification. These defects are linked to early delivery of lysosomal enzymes and fast retrieval of the vacuolar H(+)-ATPase in maturing phagosomes. We also demonstrate that EHD physically interacts with DymA. Our results indicate that EHD and DymA can associate independently with endomembranes, and yet they share identical kinetics in recruitment to phagosomes and release during phagosome maturation. Functional analysis of ehd(-), dymA(-) and double dymA(-)ehd(-) knockout strains indicate that DymA and EHD play non-redundant and independent functions in phagosome maturation. Finally, we show that the absence of EHD leads to increased tubulation of endosomes, indicating that EHD participates in the scission of endosomal tubules, as reported for DymA.
Assuntos
Dictyostelium/metabolismo , Dinaminas/metabolismo , Fagossomos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Mutação/genética , Ligação Proteica , Proteólise , Proteínas de Protozoários/química , Imagem com Lapso de TempoRESUMO
TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins.
Assuntos
Membrana Celular/metabolismo , Glicina/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Animais , Dictyostelium , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.
Assuntos
Actinas/metabolismo , Dinaminas/metabolismo , Endossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo I/metabolismo , Fagossomos/metabolismo , Proteínas de Protozoários/metabolismo , Western Blotting , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Dinaminas/genética , Endossomos/ultraestrutura , Técnicas de Inativação de Genes , Modelos Biológicos , Miosina Tipo I/genética , Fagocitose , Fagossomos/ultraestrutura , Transporte Proteico , Proteínas de Protozoários/genéticaRESUMO
Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat. When reconstituted with lipid membranes, using subtomogram averaging we determined tilted Atg18 dimer structures bridging two juxtaposed lipid membranes spaced apart by 80 Å. Moreover, lipid reconstitution experiments further delineate the contributions of Atg18's FRRG motif and the amphipathic helical extension in membrane interaction. The observed structural plasticity of Atg18's oligomeric organization and membrane binding properties provide a molecular framework for the positioning of downstream components of the autophagy machinery.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , LipídeosRESUMO
The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes.
Assuntos
Dictyostelium/metabolismo , Fagossomos/metabolismo , Animais , Imunofluorescência , Fagocitose/fisiologia , Fatores de TempoRESUMO
Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH-null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.
Assuntos
Autofagia , Dictyostelium/metabolismo , Lisossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fagocitose , Proteínas de Transporte Vesicular/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Catepsina D/metabolismo , Endossomos/metabolismo , Proteínas dos Microfilamentos/genética , Fagossomos/fisiologia , Transporte Proteico , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Transporte Vesicular/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak-Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.
Assuntos
Dictyostelium/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Dictyostelium/genética , Dictyostelium/ultraestrutura , Exocitose/genética , Exocitose/fisiologia , Genes de Protozoários , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Fenótipo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/fisiologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Actin dynamics and myosin (Myo) contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosin IK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin, and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-Pro-Arg domain that interacts with profilin and Abp1 were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A, and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.