scholarly journals Design principles of the ESCRT-III Vps24-Vps2 module

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sudeep Banjade ◽  
Yousuf H Shah ◽  
Shaogeng Tang ◽  
Scott D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Design Principles ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Lateral Association

ESCRT-III polymerization is required for all endosomal sorting complex required for transport (ESCRT)-dependent events in the cell. However, the relative contributions of the eight ESCRT-III subunits differ between each process. The minimal features of ESCRT-III proteins necessary for function and the role for the multiple ESCRT-III subunits remain unclear. To identify essential features of ESCRT-III subunits, we previously studied the polymerization mechanisms of two ESCRT-III subunits Snf7 and Vps24, identifying the association of the helix-4 region of Snf7 with the helix-1 region of Vps24 (Banjade et al., 2019a). Here, we find that mutations in the helix-1 region of another ESCRT-III subunit Vps2 can functionally replace Vps24 in Saccharomyces cerevisiae. Engineering and genetic selections revealed the required features of both subunits. Our data allow us to propose three minimal features required for ESCRT-III function – spiral formation, lateral association of the spirals through heteropolymerization, and binding to the AAA + ATPase Vps4 for dynamic remodeling.

scholarly journals Design Principles of the ESCRT-III Vps24-Vps2 Module

2020 ◽  
Author(s):  
Sudeep Banjade ◽  
Yousuf H. Shah ◽  
Shaogeng Tang ◽  
Scott D. Emr
Keyword(s):  
Design Principles ◽  
Aaa Atpase ◽  
Lateral Association

AbstractESCRT-III polymerization is required for all ESCRT-dependent events in the cell. However, the relative contributions of the eight ESCRT-III subunits differ between each process. The minimal features of ESCRT-III proteins necessary for function, and the role for the multiple ESCRT-III subunits remain unclear. To identify essential features of ESCRT-III subunits, we previously studied the polymerization mechanisms of two ESCRT-III subunits Snf7 and Vps24, identifying the association of the helix-4 region of Snf7 with the helix-1 region of Vps24 (Banjade et al., 2019). Here, we find that mutations in the helix-1 region of another ESCRT-III subunit Vps2 can functionally replace Vps24 in S. cerevisiae. Engineering and genetic selections revealed the required features of both subunits. Our data allow us to propose three minimal features required for ESCRT-III function – spiral formation, lateral association of the spirals through heteropolymerization, and binding to the AAA+ ATPase Vps4 for dynamic remodeling.

scholarly journals Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1

2007 ◽  
Vol 27 (19) ◽  
pp. 6581-6592 ◽  
Author(s):  
Brigitte Pertschy ◽  
Cosmin Saveanu ◽  
Gertrude Zisser ◽  
Alice Lebreton ◽  
Martin Tengg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Dominant Negative ◽  
Cytoplasmic Localization ◽  
Atpase Domain ◽  
Aaa Atpase ◽  
Cytoplasmic Accumulation ◽  
Functional Inactivation ◽  
Aaa Protein

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

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 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 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 Altering Sphingolipid Metabolism in Saccharomyces cerevisiae Cells Lacking the Amphiphysin Ortholog Rvs161 Reinitiates Sugar Transporter Endocytosis

2009 ◽  
Vol 8 (5) ◽  
pp. 779-789 ◽  
Author(s):  
Jeanelle Morgan ◽  
Paula McCourt ◽  
Lauren Rankin ◽  
Evelyn Swain ◽  
Lyndi M. Rice ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Sources ◽  
Transcriptional Response ◽  
Sphingolipid Metabolism ◽  
Binding Motif ◽  
Sugar Transporter ◽  
Long Chain Fatty Acid ◽  
Fatty Acid Elongase ◽  
Bar Domain ◽  
Aaa Atpase

ABSTRACT Amphiphysins are proteins thought to be involved in synaptic vesicle endocytosis. Amphiphysins share a common BAR domain, which can sense and/or bend membranes, and this function is believed to be essential for endocytosis. Saccharomyces cerevisiae cells lacking the amphiphysin ortholog Rvs161 are inviable when starved for glucose. Altering sphingolipid levels in rvs161 cells remediates this defect, but how lipid changes suppress remains to be elucidated. Here, we show that the sugar starvation-induced death of rvs161 cells extends to other fermentable sugar carbon sources, and the loss of sphingolipid metabolism suppresses these defects. In all cases, rvs161 cells respond to the starvation signal, elicit the appropriate transcriptional response, and properly localize the requisite sugar transporter(s). However, Rvs161 is required for transporter endocytosis. rvs161 cells accumulate transporters at the plasma membrane under conditions normally resulting in their endocytosis and degradation. Transporter endocytosis requires the endocytosis (endo) domain of Rvs161. Altering sphingolipid metabolism by deleting the very-long-chain fatty acid elongase SUR4 reinitiates transporter endocytosis in rvs161 and rvs161 endo − cells. The sphingolipid-dependent reinitiation of endocytosis requires the ubiquitin-regulating factors Doa1, Doa4, and Rsp5. In the case of Doa1, the phospholipase A2 family ubiquitin binding motif is dispensable. Moreover, the conserved AAA-ATPase Cdc48 and its accessory proteins Shp1 and Ufd1 are required. Finally, rvs161 cells accumulate monoubiquitin, and this defect is remediated by the loss of SUR4. These results show that defects in sphingolipid metabolism result in the reinitiation of ubiquitin-dependent sugar transporter endocytosis and suggest that this event is necessary for suppressing the nutrient starvation-induced death of rvs161 cells.

scholarly journals Sir3-Nucleosome Interactions in Spreading of Silent Chromatin in Saccharomyces cerevisiae

2008 ◽  
Vol 28 (22) ◽  
pp. 6903-6918 ◽  
Author(s):  
Johannes R. Buchberger ◽  
Megumi Onishi ◽  
Geng Li ◽  
Jan Seebacher ◽  
Adam D. Rudner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Activity ◽  
Chromatin Fiber ◽  
Silent Chromatin ◽  
Histone H4 ◽  
Histone Binding ◽  
Aaa Atpase ◽  
Binding Domains ◽  
Sir Complex

ABSTRACT Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.

scholarly journals Regulation of Vps4 ATPase activity by ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 143-145 ◽  
Author(s):  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
David J. Katzmann
Keyword(s):  
Atpase Activity ◽  
Multivesicular Body ◽  
Critical Function ◽  
Body Formation ◽  
Temporal Regulation ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Endosomal Membrane ◽  
Endosomal Membranes ◽  
Vacuolar Protein

MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

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