RESUMO
Nuclear pore assembly can go awry, but how the cell handles defective intermediates has been an ongoing question. In this issue, Lusk and colleagues describe a surveillance pathway during nuclear pore assembly and, in doing so, identify a new role for proteins that function at the endosome and plasma membrane.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Saccharomyces cerevisiae , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway's importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.
Assuntos
Síndrome de Hermanski-Pudlak , Saccharomycetales , Complexo 3 de Proteínas Adaptadoras , Animais , Endossomos , Vesículas Transportadoras , VacúolosRESUMO
Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion and the sequestration of ubiquitylated transmembrane protein cargos within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitylates ILV cargos. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in sorting of a subset of ILV cargos. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, a subunit of vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance, and disrupts vacuolar localization of Ypt7 and Vps41, two crucial mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates this fusion and rescues the ILV cargo-sorting defects in yeast that lack PI(3,5)P2 or Doa4 activity, our results suggest that the V-ATPase has a role in coordinating ILV cargo sorting with the membrane fusion machinery. This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Humanos , Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTPRESUMO
The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.
Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Ubiquitina Tiolesterase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Hidrolases/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismoRESUMO
Assembly of the endosomal sorting complex required for transport (ESCRT)-III executes the formation of intralumenal vesicles (ILVs) at endosomes. Repeated cycles of ESCRT-III function requires disassembly of the complex by Vps4, an ATPase with a microtubule interaction and trafficking (MIT) domain that binds MIT-interacting motifs (MIM1 or MIM2) in ESCRT-III subunits. We identified a putative MIT domain at the N-terminus of Doa4, which is the ubiquitin (Ub) hydrolase in Saccharomyces cerevisiae that deubiquitinates ILV cargo proteins. The Doa4 N-terminus is predicted to have the α-helical structure common to MIT domains, and it binds directly to a MIM1-like sequence in the Vps20 subunit of ESCRT-III. Disrupting this interaction does not prevent endosomal localization of Doa4 but enhances the defect in ILV cargo protein deubiquitination observed in cells lacking Bro1, which is an ESCRT-III effector protein that stimulates Doa4 catalytic activity. Deletion of the BRO1 gene (bro1Δ) blocks ILV budding, but ILV budding was rescued upon disrupting the interaction between Vps20 and Doa4. This rescue in ILV biogenesis requires Doa4 expression but is independent of its Ub hydrolase activity. Thus, binding of Vps20 to the Doa4 N-terminus inhibits a non-catalytic function of Doa4 that promotes ILV formation.
Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterase/metabolismo , Endopeptidases/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina Tiolesterase/químicaRESUMO
The endosomal sorting complexes required for transport (ESCRTs) mediate the budding of intralumenal vesicles (ILVs) at late endosomes. ESCRT dysfunction causes drastic changes in endosome morphology, which are manifested in Saccharomyces cerevisiae by the formation of aberrant endosomes known as class E compartments. Except for the absence of ILVs, the mechanistic basis for class E compartment biogenesis is unknown. We used electron microscopy to examine endosomal morphology in response to transient ESCRT inactivation and recovery in yeast expressing the temperature-sensitive mutant vps4(ts) allele. Our results show class E compartments accumulate fourfold the amount of membrane normally present at multivesicular bodies and that multivesicular bodies can form directly from class E compartments upon recovery of ESCRT function. We found class E compartment formation requires Vps21, which is orthologous to the Rab5A GTPase in metazoans that promotes fusion of endocytic vesicles with early endosomes and homotypic fusion of early endosomes with one another. We also determined that class E compartments accumulate GTP-bound Vps21 and its effector, the class C core vacuole/endosome tethering (CORVET). Ypt7, the yeast ortholog of Rab7 that in metazoans promotes fusion of late endosomes with lysosomes, also accumulates at class E compartments but without its effector, the homotypic fusion and protein sorting (HOPS), signifying that Ypt7 at class E compartments is dysfunctional. These results suggest that failure to complete Rab5-Rab7 conversion is a consequence of ESCRT dysfunction, which results in Vps21 hyperactivity that drives the class E compartment morphology. Indeed, genetic disruption of Rab conversion without ESCRT dysfunction autonomously drives the class E compartment morphology without blocking ILV budding.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Microscopia de Fluorescência , Saccharomyces cerevisiae/ultraestruturaRESUMO
Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here, we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.
Assuntos
Citosol , Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Citosol/metabolismo , Membranas Mitocondriais/metabolismo , Humanos , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , AnimaisRESUMO
Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Lipídeos , Corpos Multivesiculares , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transporte ProteicoRESUMO
Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.
Assuntos
Fosfatos , Saccharomyces cerevisiae , Animais , Potencial da Membrana Mitocondrial , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Respiração , Mamíferos/metabolismoRESUMO
Recent studies using electron microscopy, protein crystallography, classic biochemistry and novel live-cell imaging have provided numerous insights into the endocytic pathway, describing a dynamic system in which compartment morphology, molecular identity and the mechanics of cargo sorting are intimately connected. Current evidence supports a model of maturation in which the lipids, cargo proteins and Rab population at the endosome determine its competence to perform the functions of late endosomes, including the sorting of cargoes into lumenal vesicles and fusion with lysosomes.
Assuntos
Endossomos/fisiologia , Animais , Proteínas de Transporte/metabolismo , Endossomos/ultraestrutura , Humanos , Lisossomos/fisiologia , Modelos Biológicos , Vesículas Transportadoras/fisiologiaRESUMO
Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.
RESUMO
Endosomal sorting complex required for transport-III (ESCRT-III) participates in essential cellular functions, from cell division to endosome maturation. The remarkable increase of its subunit diversity through evolution may have enabled the acquisition of novel functions. Here, we characterize a novel ESCRT-III copolymer initiated by Vps60. Membrane-bound Vps60 polymers recruit Vps2, Vps24, Did2, and Ist1, as previously shown for Snf7. Snf7- and Vps60-based filaments can coexist on membranes without interacting as their polymerization and recruitment of downstream subunits remain spatially and biochemically separated. In fibroblasts, Vps60/CHMP5 and Snf7/CHMP4 are both recruited during endosomal functions and cytokinesis, but their localization is segregated and their recruitment dynamics are different. Contrary to Snf7/CHMP4, Vps60/CHMP5 is not recruited during nuclear envelope reformation. Taken together, our results show that Vps60 and Snf7 form functionally distinct ESCRT-III polymers, supporting the notion that diversification of ESCRT-III subunits through evolution is linked to the acquisition of new cellular functions.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Divisão Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/genética , Endossomos/metabolismo , Polímeros/metabolismo , HumanosRESUMO
The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/fisiologia , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Endossomos/metabolismo , Deleção de Genes , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Genéticos , Corpos Multivesiculares/metabolismo , Mutação , Fenótipo , Transporte ProteicoRESUMO
Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.
Assuntos
Citocinese/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Saccharomycetales/metabolismo , Septinas/metabolismo , Citocinese/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endocitose/genética , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Exocitose/genética , Exocitose/fisiologia , Mutação , Saccharomycetales/genética , Septinas/genéticaRESUMO
The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.
Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/fisiologia , Proteínas de Transporte/fisiologia , Endossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/ultraestrutura , Modelos Biológicos , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestruturaRESUMO
Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/enzimologia , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Microscopia de Fluorescência , Modelos Moleculares , Corpos Multivesiculares/genética , Mutação , Domínios Proteicos , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.
Assuntos
Endossomos/metabolismo , Proteínas Fúngicas/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Cristalografia por Raios X/métodos , Citoplasma/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Imunofluorescência/métodos , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/fisiologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Homologia Estrutural de Proteína , Proteínas de Transporte Vesicular/químicaRESUMO
The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.
Assuntos
Proteínas de Membrana/metabolismo , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Catálise , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Transportadoras/química , Vesículas Transportadoras/ultraestrutura , Complexos Ubiquitina-Proteína Ligase/análise , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.