MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

Lin, Tien-chen; Neuner, Annett; Flemming, Dirk; Liu, Peng; Chinen, Takumi; Jäkle, Ursula; Arkowitz, Robert A.; Schiebel, Elmar

doi:10.1083/jcb.201606092

Cited by 51 publications

(108 citation statements)

References 49 publications

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“…In Co-IP experiments, CnnT-N, CM1 and MusCM1 strongly associated with γ-Tub, whereas CnnT-N with the Δ1 mutation (CnnT-N Δ1 ) did not (Figure 4C). These results are consistent with previous findings that CM1 is essential for activating γ-TuRCs to nucleate MTs [20, 27, 28]. …”

Section: Resultssupporting

confidence: 94%

“…Our study revealed that CM1 in Cnn is essential for activating γ-TuRC to initiate MT nucleation. Consistently, CM1 of other Cnn orthologs were shown to activate γ-TuRCs [20, 27, 32]. …”

Section: Discussionmentioning

confidence: 67%

“…CnnC and CnnT both contain the CM1 domain that is important for MT nucleation [13, 20, 25–27]. To investigate CM1 function in CnnT, we generated CnnT Δ1 , which harbors the Δ1 mutation that deletes 13 conserved amino acids in CM1 [13](Figures 4A, S2A).…”

Section: Resultsmentioning

confidence: 99%

“…It is therefore remarkable that CnnT, with its simple composition of the CM1-containing γ-TuRC recruitment/binding domain fused to a mitochondrial targeting domain, is capable of organizing robust MTOCs at the mitochondrial surface without involvement of the key PCM proteins. The small protein MOZART (MZT) [35, 36] is a direct partner of CM1 [27] that also associates with GCP3 [37] and other GCP proteins [38], and cooperates with GCP6 [39]. GCP3, in particular, may directly control conformational changes of the γ-TuRC and thus regulate its MT nucleation activity [40, 41], and MZT1 appears to be a key link [27, 38].…”

Section: Discussionmentioning

confidence: 99%

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A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

et al. 2017

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Summary Non-centrosomal Microtubule Organizing Centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs have not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing, and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-TuRC, and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT. One that is necessary and sufficient to target it to mitochondria, and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation, and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.

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Section: Resultssupporting

confidence: 94%

“…Our study revealed that CM1 in Cnn is essential for activating γ-TuRC to initiate MT nucleation. Consistently, CM1 of other Cnn orthologs were shown to activate γ-TuRCs [20, 27, 32]. …”

Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

et al. 2017

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“…Various proteins such as pericentrin, NEDD1/GCP‐WD, and CDK5RAP2 have been implicated in the regulation of γTuRCs . In addition, recent studies revealed that MOZART1 (human mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1) protein may equally be involved in this regulation …”

Section: Introductionmentioning

confidence: 99%

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma‐tubulin complex

et al. 2017

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Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

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The gamma‐tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator

et al. 2021

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Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αβ-tubulin. In higher eukaryotes, de novo assembly of microtubules from αβ-tubulin is initiated by a 2 MDa multi-subunit complex, the gamma-tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer. K E Y W O R D Sgamma-tubulin ring complex, gamma-tubulin small complex, gamma-tubulin, microtubule nucleation, microtubules, recombinant gamma-tubulin ring complex Abbreviations: AAA+, adenosine triphosphatases associated with various cellular activities;Arps, actin-related proteins; CDK5RAP2, CDK5 regulatory subunit-associated protein 2; CEP192, centrosomal protein of 192 kDa; ch-TOG, colonic hepatic tumor-overexpressed gene; CM1, centrosomin motif 1; GCP, γ-TuC component protein; γ-TuCs, γ-tubulin complexes; γ-TuRC, γ-tubulin ring complex; γ-TuSC, γ-tubulin small complex; GRIP1/2, γ-tubulin ring protein 1/2; HSP90, heat shock protein 90; INO80, inositol-requiring mutant 80 complex; MZT1/2, mitotic spindle organizing protein 1/2; NEDD1, neural precursor cell expressed, developmentally down-regulated 1; NME7, nucleotide diphosphate kinase 7; PCM, pericentriolar material; PIH1D1, PIH1 domain containing 1; R2TP, Rvb1-Rvb2-Tah1-Pih1; RPAP3, RNA polymerase II associated protein 3; RUVBL1/2, RuvB-like 1/2; SPB, spindle pole body; SWR1, SWI2/SNF2-related 1 complex; XMAP215, Xenopus microtubule assembly protein with 215 kDThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

Cited by 51 publications

References 49 publications

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma‐tubulin complex

The gamma‐tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator

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