Abstract

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.

Original languageEnglish (US)
Pages (from-to)3336-3354
Number of pages19
JournalThe Journal of cell biology
Volume218
Issue number10
DOIs
StatePublished - Oct 7 2019

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Endosomal Sorting Complexes Required for Transport
Clustered Regularly Interspaced Short Palindromic Repeats
Multivesicular Bodies
Ubiquitin
Epidermal Growth Factor Receptor
Adenosine Triphosphatases
Genome
Membranes

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Takahashi, Yoshinori ; Liang, Xinwen ; Hattori, Tatsuya ; Tang, Zhenyuan ; He, Haiyan ; Chen, Han ; Liu, Xiaoming ; Abraham, Thomas ; Imamura-Kawasawa, Yuka ; Buchkovich, Nicholas J. ; Young, Megan M. ; Wang, Hong Gang. / VPS37A directs ESCRT recruitment for phagophore closure. In: The Journal of cell biology. 2019 ; Vol. 218, No. 10. pp. 3336-3354.
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abstract = "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.",
author = "Yoshinori Takahashi and Xinwen Liang and Tatsuya Hattori and Zhenyuan Tang and Haiyan He and Han Chen and Xiaoming Liu and Thomas Abraham and Yuka Imamura-Kawasawa and Buchkovich, {Nicholas J.} and Young, {Megan M.} and Wang, {Hong Gang}",
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VPS37A directs ESCRT recruitment for phagophore closure. / Takahashi, Yoshinori; Liang, Xinwen; Hattori, Tatsuya; Tang, Zhenyuan; He, Haiyan; Chen, Han; Liu, Xiaoming; Abraham, Thomas; Imamura-Kawasawa, Yuka; Buchkovich, Nicholas J.; Young, Megan M.; Wang, Hong Gang.

In: The Journal of cell biology, Vol. 218, No. 10, 07.10.2019, p. 3336-3354.

Research output: Contribution to journalArticle

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T1 - VPS37A directs ESCRT recruitment for phagophore closure

AU - Takahashi, Yoshinori

AU - Liang, Xinwen

AU - Hattori, Tatsuya

AU - Tang, Zhenyuan

AU - He, Haiyan

AU - Chen, Han

AU - Liu, Xiaoming

AU - Abraham, Thomas

AU - Imamura-Kawasawa, Yuka

AU - Buchkovich, Nicholas J.

AU - Young, Megan M.

AU - Wang, Hong Gang

PY - 2019/10/7

Y1 - 2019/10/7

N2 - 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.

AB - 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.

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