scholarly journals ATP-dependent force generation and membrane scission by ESCRT-III and Vps4

Science ◽  
2018 ◽  
Vol 362 (6421) ◽  
pp. 1423-1428 ◽  
Author(s):  
Johannes Schöneberg ◽  
Mark Remec Pavlin ◽  
Shannon Yan ◽  
Maurizio Righini ◽  
Il-Hyung Lee ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Adenosine Triphosphatase ◽  
Atp Release ◽  
Force Generation ◽  
Giant Vesicles ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Membrane Nanotubes ◽  
Membrane Scission ◽  
Correct Topology

The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 and Vps4 puncta within nanotubes whose presence followed ATP release, correlated with force generation and nanotube constriction, and preceded scission. These observations directly verify long-standing predictions that ATP-hydrolyzing assemblies of ESCRT-III and Vps4 sever membranes.

scholarly journals ATP-dependent force generation and membrane scission by ESCRT-III and Vps4

2018 ◽  
Author(s):  
Johannes Schöneberg ◽  
Shannon Yan ◽  
Maurizio Righini ◽  
Mark Remec Pavlin ◽  
Il-Hyung Lee ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Atp Release ◽  
Force Production ◽  
Force Generation ◽  
Membrane Insertion ◽  
Giant Vesicles ◽  
Membrane Nanotubes ◽  
Membrane Scission ◽  
Correct Topology

AbstractThe ESCRTs catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated a minimal ESCRT module consisting of ESCRT-III subunits Snf7, Vps24, and Vps2, and the AAA+ ATPase Vps4 such that membrane nanotubes reflecting the correct topology of scission could be pulled from giant vesicles. Upon ATP release by photo-uncaging, this system was capable of generating forces within the nanotubes in a manner dependent upon Vps4 catalytic activity, Vps4 coupling to the ESCRT-III proteins, and membrane insertion by Snf7. At physiological concentrations, single scission events were observed that correlated with forces of ~6 pN, verifying predictions that ESCRTs are capable of exerting forces on membranes. Imaging of scission with subsecond resolution revealed Snf7 puncta at the sites of membrane cutting, directly verifying longstanding predictions for the ESCRT scission mechanism.One Sentence SummaryESCRT-III and Vps4 were reconstituted from within the interior of nanotubes pulled from giant vesicles, revealing that this machinery couples ATP-dependent force production for membrane scission.

scholarly journals Rules for the self-assembly of ESCRT-III on endosomes

2021 ◽  
Author(s):  
Simon Sprenger ◽  
Simona M. Migliano ◽  
Florian Oleschko ◽  
Marvin Kobald ◽  
Michael Hess ◽  
...  
Keyword(s):  
Self Assembly ◽  
Hydrophobic Interactions ◽  
The Self ◽  
Amino Acid Residues ◽  
Lateral Expansion ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Charged Amino Acids ◽  
Membrane Budding ◽  
Positively Charged

ABSTRACTThe endosomal sorting complexes required for transport (ESCRT) mediate various membrane remodeling processes in cells by mechanism that are incompletely understood. Here we combined genetic experiments in budding yeast with site-specific cross-linking to identify rules that govern the self-assembly of individual ESCRT-III proteins into functional ESCRT-III complexes on endosomes. Together with current structural models of ESCRT-III, our findings suggest that, once nucleated, the growing Snf7 protofilament seeds the lateral co-assembly of a Vps24 - Vps2 heterofilament. Both Vps24 and Vps2 use positively charged amino acid residues in their helices α1 to interact with negatively charged amino acids in helix α4 of Snf7 subunits of the protofilament. In the Vps24 - Vps2 heterofilament, the two subunits alternate and interact with each other using hydrophobic interactions between helices α2/α3. The co-assembly of the Vps24 - Vps2 heterofilament restricts the lateral expansion of Snf7 protofilaments and leads the immediate recruitment of the AAA-ATPase Vps4. This self-assembly process of three ESCRT-III subunits results in the formation of a Snf7 protofilament and the co-assembly of a Vps24 - Vps2 heterofilament. This sets the stage for Vps4 recruitment and the subsequent ATP-driven dynamic self-organization of ESCRT-III / Vps4 assemblies and the ensuing membrane budding and scission events.

scholarly journals Membrane Nanotubes Increase the Robustness of Giant Vesicles

ACS Nano ◽  
2018 ◽  
Vol 12 (5) ◽  
pp. 4478-4485 ◽  
Author(s):  
Tripta Bhatia ◽  
Jaime Agudo-Canalejo ◽  
Rumiana Dimova ◽  
Reinhard Lipowsky
Keyword(s):  
Giant Vesicles ◽  
Membrane Nanotubes

scholarly journals Structure and mechanism of the ESCRT pathway AAA+ ATPase Vps4

2019 ◽  
Vol 47 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Han Han ◽  
Christopher P. Hill
Keyword(s):  
Cell Biology ◽  
Cellular Membrane ◽  
Structural Features ◽  
Transport Pathways ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Protein Substrates ◽  
Membrane Fission ◽  
Aaa Atpases ◽  
Equivalent Mechanism

Abstract The progression of ESCRT (Endosomal Sorting Complexes Required for Transport) pathways, which mediate numerous cellular membrane fission events, is driven by the enzyme Vps4. Understanding of Vps4 mechanism is, therefore, of fundamental importance in its own right and, moreover, it is highly relevant to the understanding of many related AAA+ ATPases that function in multiple facets of cell biology. Vps4 unfolds its ESCRT-III protein substrates by translocating them through its central hexameric pore, thereby driving membrane fission and recycling of ESCRT-III subunits. This mini-review focuses on recent advances in Vps4 structure and mechanism, including ideas about how Vps4 translocates and unfolds ESCRT-III subunits. Related AAA+ ATPases that share structural features with Vps4 and likely utilize an equivalent mechanism are also discussed.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

scholarly journals Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
David Oriola ◽  
Sophie Roth ◽  
Marileen Dogterom ◽  
Jaume Casademunt
Keyword(s):  
Motor Performance ◽  
Vesicular Transport ◽  
Force Generation ◽  
Theoretical Studies ◽  
Giant Vesicles ◽  
Traffic Conditions ◽  
Self Organized ◽  
Mechanochemical Cycle ◽  
Axonal Trafficking

Abstract The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochemical cycle. However, recent theoretical studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to extract membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to extract tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helices. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvreing capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.

scholarly journals Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1

2006 ◽  
Vol 172 (5) ◽  
pp. 705-717 ◽  
Author(s):  
Ishara Azmi ◽  
Brian Davies ◽  
Christian Dimaano ◽  
Johanna Payne ◽  
Debra Eckert ◽  
...  
Keyword(s):  
Protein Composition ◽  
Surface Protein ◽  
Multivesicular Body ◽  
Proper Function ◽  
Cellular Functions ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Evolutionarily Conserved

In eukaryotes, the multivesicular body (MVB) sorting pathway plays an essential role in regulating cell surface protein composition, thereby impacting numerous cellular functions. Vps4, an ATPase associated with a variety of cellular activities, is required late in the MVB sorting reaction to dissociate the endosomal sorting complex required for transport (ESCRT), a requisite for proper function of this pathway. However, regulation of Vps4 function is not understood. We characterize Vta1 as a positive regulator of Vps4 both in vivo and in vitro. Vta1 promotes proper assembly of Vps4 and stimulates its ATPase activity through the conserved Vta1/SBP1/LIP5 region present in Vta1 homologues across evolution, including human SBP1 and Arabidopsis thaliana LIP5. These results suggest an evolutionarily conserved mechanism through which the disassembly of the ESCRT proteins, and thereby MVB sorting, is regulated by the Vta1/SBP1/LIP5 proteins.

scholarly journals CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway

2006 ◽  
Vol 400 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Mio Horii ◽  
Hideki Shibata ◽  
Ryota Kobayashi ◽  
Keiichi Katoh ◽  
Chiharu Yorikawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Positive Interaction ◽  
Leukaemia Virus ◽  
Gag Protein ◽  
Endosomal Sorting ◽  
Aaa Atpase

All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1–CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG–CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG–CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b–GFP or GFP–Vps4BE235Q, a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP–CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP–CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting 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 Effect of removal of calcium-activated adenosine triphosphatase from rat mast cells by treatment with sodium glycocholate

1977 ◽  
Vol 168 (3) ◽  
pp. 583-585 ◽  
Author(s):  
P H Cooper ◽  
D R Stanworth
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Adenosine Triphosphatase ◽  
External Surface ◽  
Atp Release ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Sodium Glycocholate ◽  
Rat Mast Cells

Sodium glycocholate was shown to remove a Ca2+-activated adenosine triphosphatase from the external surface of the rat mast cell without causing lysis. Sensitized mast cells pretreated with sodium glycocholate showed a decrease in histamine-releasing capacity when triggered with antigen, Synacthen and ATP. Release induced by calcium ionophore A23187 was unaffected.

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