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Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate pote...

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Autores principales: Tozawa, Takafumi, Matsunaga, Kohichi, Izumi, Tetsuro, Shigehisa, Naotake, Uekita, Takamasa, Taoka, Masato, Ichimura, Tohru
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355226/
https://www.ncbi.nlm.nih.gov/pubmed/35930602
http://dx.doi.org/10.1371/journal.pone.0272700
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author Tozawa, Takafumi
Matsunaga, Kohichi
Izumi, Tetsuro
Shigehisa, Naotake
Uekita, Takamasa
Taoka, Masato
Ichimura, Tohru
author_facet Tozawa, Takafumi
Matsunaga, Kohichi
Izumi, Tetsuro
Shigehisa, Naotake
Uekita, Takamasa
Taoka, Masato
Ichimura, Tohru
author_sort Tozawa, Takafumi
collection PubMed
description Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.
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spelling pubmed-93552262022-08-06 Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies Tozawa, Takafumi Matsunaga, Kohichi Izumi, Tetsuro Shigehisa, Naotake Uekita, Takamasa Taoka, Masato Ichimura, Tohru PLoS One Research Article Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination. Public Library of Science 2022-08-05 /pmc/articles/PMC9355226/ /pubmed/35930602 http://dx.doi.org/10.1371/journal.pone.0272700 Text en © 2022 Tozawa et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Tozawa, Takafumi
Matsunaga, Kohichi
Izumi, Tetsuro
Shigehisa, Naotake
Uekita, Takamasa
Taoka, Masato
Ichimura, Tohru
Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title_full Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title_fullStr Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title_full_unstemmed Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title_short Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies
title_sort ubiquitination-coupled liquid phase separation regulates the accumulation of the trim family of ubiquitin ligases into cytoplasmic bodies
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9355226/
https://www.ncbi.nlm.nih.gov/pubmed/35930602
http://dx.doi.org/10.1371/journal.pone.0272700
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