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Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1

Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [1, 2, 3, 4, 5, 6, 7]. Additional conserved regulators of the...

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Autores principales: Leong, Su Ling, Lynch, Eric M., Zou, Juan, Tay, Ye Dee, Borek, Weronika E., Tuijtel, Maarten W., Rappsilber, Juri, Sawin, Kenneth E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cell Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616311/
https://www.ncbi.nlm.nih.gov/pubmed/31287970
http://dx.doi.org/10.1016/j.cub.2019.05.058
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author Leong, Su Ling
Lynch, Eric M.
Zou, Juan
Tay, Ye Dee
Borek, Weronika E.
Tuijtel, Maarten W.
Rappsilber, Juri
Sawin, Kenneth E.
author_facet Leong, Su Ling
Lynch, Eric M.
Zou, Juan
Tay, Ye Dee
Borek, Weronika E.
Tuijtel, Maarten W.
Rappsilber, Juri
Sawin, Kenneth E.
author_sort Leong, Su Ling
collection PubMed
description Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [1, 2, 3, 4, 5, 6, 7]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [8, 9, 10, 11, 12, 13] and proteins containing a Centrosomin Motif 1 (CM1) domain [10, 14, 15, 16, 17, 18, 19]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe. The S. pombe CM1 protein Mto1 recruits the γ-TuC to microtubule-organizing centers (MTOCs) [14, 20, 21, 22], and analysis of Mto1[bonsai], a truncated version of Mto1 that cannot localize to MTOCs, has shown that Mto1 also has a role in γ-TuC activation [23]. S. pombe Mzt1 interacts with γ-TuSC and is essential for γ-TuC function and localization to MTOCs [11, 12]. However, the mechanisms by which Mzt1 functions remain unclear. Here we describe reconstitution of MT nucleation using purified recombinant Mto1[bonsai], the Mto1 partner protein Mto2, γ-TuSC, and Mzt1. Multiple copies of the six proteins involved coassemble to form a 34-40S ring-like “MGM” holocomplex that is a potent MT nucleator in vitro. Using purified MGM and subcomplexes, we investigate the role of Mzt1 in MT nucleation. Our results suggest that Mzt1 is critical to stabilize Alp6, the S. pombe homolog of human γ-TuSC protein GCP3, in an “interaction-competent” form within the γ-TuSC. This is essential for MGM to become a functional nucleator.
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spelling pubmed-66163112019-07-22 Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1 Leong, Su Ling Lynch, Eric M. Zou, Juan Tay, Ye Dee Borek, Weronika E. Tuijtel, Maarten W. Rappsilber, Juri Sawin, Kenneth E. Curr Biol Article Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [1, 2, 3, 4, 5, 6, 7]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [8, 9, 10, 11, 12, 13] and proteins containing a Centrosomin Motif 1 (CM1) domain [10, 14, 15, 16, 17, 18, 19]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe. The S. pombe CM1 protein Mto1 recruits the γ-TuC to microtubule-organizing centers (MTOCs) [14, 20, 21, 22], and analysis of Mto1[bonsai], a truncated version of Mto1 that cannot localize to MTOCs, has shown that Mto1 also has a role in γ-TuC activation [23]. S. pombe Mzt1 interacts with γ-TuSC and is essential for γ-TuC function and localization to MTOCs [11, 12]. However, the mechanisms by which Mzt1 functions remain unclear. Here we describe reconstitution of MT nucleation using purified recombinant Mto1[bonsai], the Mto1 partner protein Mto2, γ-TuSC, and Mzt1. Multiple copies of the six proteins involved coassemble to form a 34-40S ring-like “MGM” holocomplex that is a potent MT nucleator in vitro. Using purified MGM and subcomplexes, we investigate the role of Mzt1 in MT nucleation. Our results suggest that Mzt1 is critical to stabilize Alp6, the S. pombe homolog of human γ-TuSC protein GCP3, in an “interaction-competent” form within the γ-TuSC. This is essential for MGM to become a functional nucleator. Cell Press 2019-07-08 /pmc/articles/PMC6616311/ /pubmed/31287970 http://dx.doi.org/10.1016/j.cub.2019.05.058 Text en © 2019 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Leong, Su Ling
Lynch, Eric M.
Zou, Juan
Tay, Ye Dee
Borek, Weronika E.
Tuijtel, Maarten W.
Rappsilber, Juri
Sawin, Kenneth E.
Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title_full Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title_fullStr Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title_full_unstemmed Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title_short Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1
title_sort reconstitution of microtubule nucleation in vitro reveals novel roles for mzt1
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616311/
https://www.ncbi.nlm.nih.gov/pubmed/31287970
http://dx.doi.org/10.1016/j.cub.2019.05.058
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