VPS37A directs ESCRT recruitment for phagophore closure

Takahashi, Yoshinori; Liang, Xinwen; Hattori, Tatsuya; Tang, Zifan; He, Haiyan; Chen, Han; Liu, Xiaoming; Abraham, Thomas; Imamura‐Kawasawa, Yuka; Buchkovich, Nicholas J.; Young, Michelle; Wang, Hong Gang

doi:10.1083/jcb.201902170

Cited by 95 publications

(120 citation statements)

References 51 publications

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“…In regard to the similar topology for the invagination of the internal vesicles from the outer membrane, the ESCRT complex would be a good membrane scission candidate for sealing the autophagosome membrane ( Figure 1C). Indeed, in most ESCRT mutants, abnormal autophagosome structures are often detected in the cytosol (Takahashi et al, 2018(Takahashi et al, , 2019Zhou et al, 2019). Recently, using a novel method to distinguish the phagophore and nascent autophagosome by labeling LC3 with permeable and impermeable dyes, it was shown that an ESCRT-III component CHMP2A and an AAA-ATPase vacuolar protein sorting-associated 4 (VPS4) translocate to the phagophore during autophagy (Takahashi et al, 2018).…”

Section: Autophagosome Closurementioning

confidence: 99%

Recent Advances in Membrane Shaping for Plant Autophagosome Biogenesis

2020

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Autophagy is an intracellular degradation process, which is highly conserved in eukaryotes. During this process, unwanted cytosolic constituents are sequestered and delivered into the vacuole/lysosome by a double-membrane organelle known as an autophagosome. The autophagosome initiates from a membrane sac named the phagophore, and after phagophore expansion and closure, the outer membrane fuses with the vacuole/lysosome to release the autophagic body into the vacuole. Membrane sources derived from the endomembrane system (e.g., Endoplasmic Reticulum, Golgi and endosome) have been implicated to contribute to autophagosome in different steps (initiation, expansion or maturation). Therefore, coordination between the autophagy-related (ATG) proteins and membrane tethers from the endomembrane system is required during autophagosome biogenesis. In this review, we will update recent findings with a focus on comparing the selected core ATG complexes and the endomembrane tethering machineries for shaping the autophagosome membrane in yeast, mammal, and plant systems.

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Section: Autophagosome Closurementioning

confidence: 99%

Recent Advances in Membrane Shaping for Plant Autophagosome Biogenesis

2020

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“…ESCRTs mediate membrane remodeling and scission in various processes, including multivesicular endosome formation and cytokinesis (Schöneberg et al, 2016;McCullough et al, 2018). In autophagy, ESCRTs facilitate autophagosome closure, which involves membrane budding away from the cytosol and is therefore topologically equivalent to other ESCRT-mediated processes (Hurley, 2015;Spitzer et al, 2015;Takahashi et al, 2019;Zhou et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast

et al. 2019

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ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro-and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

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“…Homozygous mutation of the equivalent residue within VPS37A to asparagine has been linked with complex hereditary spastic paraparesis (CHSP) in multiple individuals 51 . It is interesting that VPS37A is the ortholog uniquely involved in autophagosome closure 49 , but it is currently whether or how this role is connected to the disease phenotype. In ESCRT-I tetrameric complexes are gradually recruited to Gag 62 .…”

Section: Discussionmentioning

confidence: 99%

“…To examine the importance of the helical interface in ESCRT-I assembly in vivo, we examined the role of VPS28-mediated ring assembly in ESCRT recruitment to autophagosomes. HAtagged VPS28 TM and control wild-type VPS28 (VPS28 WT) were lentivirally transduced into U-2 OS human osteosarcoma cells in which endogenous VPS37A is replaced by GFP-VPS37A 49 . Consistent with in vitro data, exogenously-introduced VPS28 displaced nearly all endogenous VPS28 and successfully incorporated into the ESCRT-I complex regardless of the mutations (Fig.…”

Section: The Helical Interface Of Vps28 Stabilizes Escrt-i Oligomerizmentioning

confidence: 99%

“…To induce autophagy, cells were rinsed three times with Dulbecco's Phosphate Buffered Saline (DPBS) and incubated with amino acid-free DMEM (Wako, 048-33575) for 3 h. HT-LC3 autophagosome completion assay was performed as described previously 49 . For immunoblotting-based autophagic flux assay, cell lysates were prepared in radioimmunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate, 5 mM EDTA, pH 8.0) containing protease inhibitors and subjected to SDS-PAGE followed by immunoblotting with antibodies against LC3, p62 and β-ACTIN.…”

Section: Autophagy Assaysmentioning

confidence: 99%

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A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission

Flower

Takahashi

Hudait

et al. 2020

Preprint

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The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, MVB biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101:VPS28:VPS37B:MVB12A was determined, revealing an ESCRT-I helical assembly with a 12 molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse grained simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding, and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor, but has an essential scaffolding and mechanical role in its own right.The Endosomal Sorting Complexes Required for Transport (ESCRTs) catalyze so-called "reverse topology" membrane scission events, defined as those in which budding is directed away from the cytosol 1,2 . Such events include, but are not limited to, multivesicular body (MVB) biogenesis, HIV-1 particle egress 3,4 ; cytokinesis 5 ; autophagosome closure 6-8 ; and nuclear envelope (NE) reformation 9,10 . The core ESCRT machinery consists of the ESCRT-I, ESCRT-II and ESCRT-III complexes, ALIX, and the AAA + ATPase VPS4. ESCRT-III and VPS4 are considered to be the minimal machinery for membrane scission, in that they can carry this reaction out in vitro in the absence of the upstream components 11 . The ESCRT-IIIs are a family of small alpha helical proteins that can self-assemble into membrane-associated, homoand hetero-polymeric structures. These polymeric assemblies can adopt many different morphologies, including cones 12 , coils 13 , flat spirals 13-17 , and helical tubes 12,14,18,19 . The subunits within ESCRT-III polymers are exchanged with cytosolic monomers by VPS4 20 , leading to scission 11 . Viral proteins and host cargoes are recognized by the upstream ESCRT-I complex 21-24 or ALIX 25,26 . ESCRT-I in turn recruits ESCRT-II and then CHMP6, the most upstream of the ESCRT-III proteins. ALIX, and its paralog HD-PTP, provide a parallel route for ESCRT-III recruitment via CHMP4 27-30 . It is still unclear how ESCRT-I/-II and ALIX orchestrate ESCRT-III polymerization for scission. Each of these complexes are rigid and elongated, and their physical dimensions approximate those of the membrane necks with which they function. A central question is whether ESCRT-I (and other upstream ESCRTs) merely bridges cargo and the downstream ESCRTs, or whether it has a more specific and active mechanical role in scaffolding membrane remodeling and scission. The critical Pro-Thr/Ser-Ala-Pro (PTAP) motif of HIV-1 Gag is recognized by the ubiquitin E2 variant (UEV) domain of TSG101 31-34 . Human ESCRT-I...

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VPS37A directs ESCRT recruitment for phagophore closure

Cited by 95 publications

References 51 publications

Recent Advances in Membrane Shaping for Plant Autophagosome Biogenesis

Recent Advances in Membrane Shaping for Plant Autophagosome Biogenesis

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast

A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission

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