scholarly journals Structure and assembly of ESCRT-III helical Vps24 filaments

2020 ◽  
Vol 6 (34) ◽  
pp. eaba4897 ◽  
Author(s):  
Stefan T. Huber ◽  
Siavash Mostafavi ◽  
Simon A. Mortensen ◽  
Carsten Sachse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mixed Type ◽  
Multivesicular Body ◽  
Cellular Membrane ◽  
Membrane Remodeling ◽  
Tubular Structures ◽  
Geometric Properties ◽  
Open Conformation ◽  
Assembly Structure ◽  
Polymeric Structures

ESCRT-III proteins mediate a range of cellular membrane remodeling activities such as multivesicular body biogenesis, cytokinesis, and viral release. Critical to these processes is the assembly of ESCRT-III subunits into polymeric structures. In this study, we determined the cryo-EM structure of a helical assembly of Saccharomyces cerevisiae Vps24 at 3.2-Å resolution and found that Vps24 adopts an elongated open conformation. Vps24 forms a domain-swapped dimer extended into protofilaments that associate into a double-stranded apolar filament. We demonstrate that, upon binding negatively charged lipids, Vps24 homopolymer filaments undergo partial disassembly into shorter filament fragments and oligomers. Upon the addition of Vps24, Vps2, and Snf7, liposomes are deformed into neck and tubular structures by an ESCRT-III heteropolymer coat. The filamentous Vps24 homopolymer assembly structure and interaction studies reveal how Vps24 could introduce unique geometric properties to mixed-type ESCRT-III heteropolymers and contribute to the process of membrane scission events.

scholarly journals Interaction Maps of the Saccharomyces cerevisiae ESCRT-III Protein Snf7

2013 ◽  
Vol 12 (11) ◽  
pp. 1538-1546 ◽  
Author(s):  
Barbara Sciskala ◽  
Ralf Kölling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Interaction Network ◽  
Multivesicular Body ◽  
Binding Motifs ◽  
Content Type ◽  
Endosomal Membrane ◽  
Binding Partners ◽  
First Time ◽  
Complementary Binding

ABSTRACT The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S -transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.

Structure and function of ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 156-160 ◽  
Author(s):  
Suman Lata ◽  
Guy Schoehn ◽  
Julianna Solomons ◽  
Ricardo Pires ◽  
Heinrich G. Göttlinger ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Tubular Structures ◽  
Body Protein ◽  
Enveloped Viruses ◽  
Endosomal Sorting ◽  
Vacuolar Protein ◽  
And Function ◽  
Endosomal Vesicles

ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A–CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.

scholarly journals Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

2004 ◽  
Vol 166 (5) ◽  
pp. 717-729 ◽  
Author(s):  
Natalie Luhtala ◽  
Greg Odorizzi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Multivesicular Body ◽  
Integral Membrane Proteins ◽  
Surface Receptors ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Late Step ◽  
Endosomal Membranes ◽  
Multivesicular Endosomes ◽  
Mutant Cells

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.

scholarly journals Timing of ESCRT-III protein recruitment and membrane scission during HIV-1 assembly

2018 ◽  
Author(s):  
Daniel S. Johnson ◽  
Marina Bleck ◽  
Sanford M. Simon
Keyword(s):  
Multivesicular Body ◽  
Cellular Membrane ◽  
Membrane Curvature ◽  
Gag Protein ◽  
Endosomal Sorting ◽  
Membrane Scission ◽  
Nuclear Envelope Formation ◽  
Protein Recruitment ◽  
Late Domains ◽  
Hiv 1

The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ∼20 seconds before scission. These observations suggest ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-III and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.

scholarly journals The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

2021 ◽  
Author(s):  
Cyrine Bentaleb ◽  
Kévin Hervouet ◽  
Claire Montpellier ◽  
Charline Camuzet ◽  
Julien Burlaud-Gaillard ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Cellular Membrane ◽  
Tubular Structures ◽  
Endocytic Recycling ◽  
Orf3 Protein ◽  
Positive Strand Rna ◽  
Viral Factory ◽  
The Impact

Background & Aims: Although Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms. Methods: We generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC). Results: One of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC. Conclusions: Our study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

scholarly journals Extracellular pH and high concentration of potassium regulate the primary necrosis in the yeast Saccharomyces cerevisiae.

2021 ◽  
Author(s):  
Victoria Bidiuk ◽  
Alexander Alexandrov ◽  
Airat Valiakhmetov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Yeast Cell ◽  
Gene Mutations ◽  
Cellular Membrane ◽  
Extracellular Ph ◽  
Specific Gene ◽  
High Concentration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Neutral Ph

Abstract Extracellular pH has a significant impact on the physiology of the yeast cell, but its role in cell death has not been thoroughly investigated. We studied the effect of extracellular pH on the development of primary necrosis in Saccharomyces cerevisiae yeast under two general conditions leading to cell death. The first is sugar induced cell death (SICD), and the second is death caused by several specific gene deletions, which have been recently identified in a systematic screen. It was shown that in both cases, primary necrosis is suppressed at neutral pH. SICD was also inhibited by the protonophore dinitrophenol (DNP) and 150 mM extracellular K+, with the latter condition also benefiting survival of cell dying due to gene mutations. Thus, we show that neutral pH can suppress different types of primary necrosis. We suggest that changes to the cellular membrane potential can play a central role in yeast cell death.

scholarly journals Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

2020 ◽  
Author(s):  
Moona Huttunen ◽  
Artur Yakimovich ◽  
Ian J. White ◽  
Janos Kriston-Vizi ◽  
Juan Martin-Serrano ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Multivesicular Body ◽  
Cellular Membrane ◽  
Double Membrane ◽  
Endosomal Sorting ◽  
A Cell ◽  
Infected Cells ◽  
Membrane Bound ◽  
Virus Egress

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

scholarly journals Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae

2018 ◽  
Vol 74 (10) ◽  
pp. 617-624 ◽  
Author(s):  
Shu Moriyama ◽  
Kazuya Nishio ◽  
Tsunehiro Mizushima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Malate Dehydrogenase ◽  
Ternary Complex ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Tricarboxylic Acid ◽  
Mitochondrial Enzyme ◽  
Open Conformation ◽  
Metabolism Enzyme ◽  
The Glyoxylate Cycle

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3–NAD+ complex and the MDH3–NAD+–OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

scholarly journals Structure, Folding and Stability of Nucleoside Diphosphate Kinases

2020 ◽  
Vol 21 (18) ◽  
pp. 6779
Author(s):  
Florian Georgescauld ◽  
Yuyu Song ◽  
Alain Dautant
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Membrane Remodeling ◽  
Oligomeric Proteins ◽  
X Ray ◽  
X Ray Crystallography ◽  
Assembly Structure ◽  
Nucleoside Diphosphate Kinases ◽  
Cytoskeleton Dynamics

Nucleoside diphosphate kinases (NDPK) are oligomeric proteins involved in the synthesis of nucleoside triphosphates. Their tridimensional structure has been solved by X-ray crystallography and shows that individual subunits present a conserved ferredoxin fold of about 140 residues in prokaryotes, archaea, eukaryotes and viruses. Monomers are functionally independent from each other inside NDPK complexes and the nucleoside kinase catalytic mechanism involves transient phosphorylation of the conserved catalytic histidine. To be active, monomers must assemble into conserved head to tail dimers, which further assemble into hexamers or tetramers. The interfaces between these oligomeric states are very different but, surprisingly, the assembly structure barely affects the catalytic efficiency of the enzyme. While it has been shown that assembly into hexamers induces full formation of the catalytic site and stabilizes the complex, it is unclear why assembly into tetramers is required for function. Several additional activities have been revealed for NDPK, especially in metastasis spreading, cytoskeleton dynamics, DNA binding and membrane remodeling. However, we still lack the high resolution structural data of NDPK in complex with different partners, which is necessary for deciphering the mechanism of these diverse functions. In this review we discuss advances in the structure, folding and stability of NDPKs.

scholarly journals Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Chun-Che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ubiquitin Binding ◽  
Stimulation Of ◽  
Cargo Sorting

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.

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