The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.
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1 March 2021
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January 26 2021
Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function
Michal Wieczorek
,
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
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Shih-Chieh Ti
,
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
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Linas Urnavicius
,
Linas Urnavicius
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
2
Laboratory of Cell Biology, The Rockefeller University, New York, NY
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Kelly R. Molloy
,
Kelly R. Molloy
3
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY
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Amol Aher
,
Amol Aher
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
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Brian T. Chait
,
Brian T. Chait
3
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY
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Tarun M. Kapoor
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
Correspondence to Tarun M. Kapoor: kapoor@rockefeller.edu
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Michal Wieczorek
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
Shih-Chieh Ti
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
Linas Urnavicius
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
2
Laboratory of Cell Biology, The Rockefeller University, New York, NY
Kelly R. Molloy
3
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY
Amol Aher
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
Brian T. Chait
3
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY
Tarun M. Kapoor
1
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY
Correspondence to Tarun M. Kapoor: kapoor@rockefeller.edu
*
M. Wieczorek and S.-C. Ti contributed equally to this paper.
S.-C. Ti’s present address is School of Biomedical Sciences, Faculty of Medicine, University of Hong Kong, Hong Kong
Received:
September 21 2020
Revision Received:
December 10 2020
Accepted:
January 04 2021
Online Issn: 1540-8140
Print Issn: 0021-9525
Funding:
Human Frontier Science Program
(LT000025/18-L1)
National Institutes of Health
(NO AWARD)
Rockefeller University
(NO AWARD)
© 2021 Wieczorek et al.
2021
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Cell Biol (2021) 220 (3): e202009146.
Article history
Received:
September 21 2020
Revision Received:
December 10 2020
Accepted:
January 04 2021
Connected Content
Commentary
Microtubule nucleation without a ring?
Citation
Michal Wieczorek, Shih-Chieh Ti, Linas Urnavicius, Kelly R. Molloy, Amol Aher, Brian T. Chait, Tarun M. Kapoor; Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function. J Cell Biol 1 March 2021; 220 (3): e202009146. doi: https://doi.org/10.1083/jcb.202009146
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