Insights into the assembly and activation of the microtubule nucleator γ-TuRC

Liu, Peng; Župa, Erik; Neuner, Annett; Böhler, Anna; Loerke, Justus; Flemming, Dirk; Ruppert, Thomas; Rudack, Till; Peter, Christoph; Spahn, C.M.T.; Gruß, Oliver J.; Pfeffer, Stefan; Schiebel, Elmar

doi:10.1038/s41586-019-1896-6

Cited by 126 publications

(299 citation statements)

References 42 publications

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“…We demonstrate that GCPs 4, 5, and 6 form a stable sub-complex that permits the association with γTuSCs into a functional γTuRC. Within the sub-complex, the stoichiometric ratio of these GCPs matches the values found in recently reported structures of native γTuRCs (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). We find that GCPs 2 and 3 are also present in our preparations of the sub-complex, but at a molar ratio equivalent to half-a-γTuSC.…”

Section: Discussionsupporting

confidence: 87%

“…Since Wieczorek et al (2020) and Liu et al (2019) suggested the existence of γTuSC-like structures, containing pairs of GCPs 4/5 or GCPs 4/6, we tested how the depletion of individual GCPs 4, 5, or 6 affected the composition of the GCP4/5/6 sub-complex, and whether the proposed γTuSC-like structures did indeed exist. We depleted each of these three GCPs individually by RNAi, which resulted in each case in the loss of γTuRCs and in the accumulation of γTuSCs, as seen in sucrose gradients (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…GCPs 4, 5, and 6 are essential for the assembly and/or for the stabilization of γTuRCs, since depletion of either component causes a reduction of γTuRCs both at the centrosome and in the cytoplasm (Izumi et al, 2008; Bahtz et al, 2012; Scheidecker et al, 2015; Farache et al, 2016; Cota et al, 2017). Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Our results show that they form a nucleus promoting the stable assembly of the 2MDa complex.…”

Section: Discussionmentioning

confidence: 99%

“…Because these proteins are present only in one or two copies per complex (Murphy et al, 2001; Choi et al, 2010), and because the rescue experiments with chimeras suggest that GCPs 4, 5, 6 occupy non-random positions, their localisation within the complex is of particular interest. During the course of this work, three studies have described the structure of native γTuRCs by cry-electron microscopy, and have found a lateral association of four γTuSCs, bound to a lateral array of GCP4/GCP5/GCP4/GCP6, to which an additional γTuSC was associated (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Altogether, this has raised the question whether GCPs 4, 5, 6 form assembly intermediates equivalent to γTuSCs.…”

Section: Introductionmentioning

confidence: 99%

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A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Merdes

Haren

Farache

et al. 2020

Preprint

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Abstractγ-tubulin is a major protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (gamma-tubulin complex proteins): γ-tubulin small complexes (γTuSCs), containing γ-tubulin and GCPs 2 and 3, and γ-tubulin ring complexes (γTuRCs), containing multiple γTuSCs, in addition to GCPs 4, 5, and 6. Whereas the structure and assembly properties of γTuSCs have been intensively studied, little is known about the assembly of γTuRCs, and about the specific roles of GCPs 4, 5, and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt-resistant sub-complex within the γTuRC that assembles independently of the presence of γTuSCs. Incubation of this sub-complex with cytoplasmic extracts containing γTuSCs leads to the reconstitution of full-sized γTuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the γTuRC.Summary statementγ-tubulin ring complexes are templates for microtubule nucleation, composed of γ-tubulin and GCP proteins. GCPs 4, 5, 6 form a stable sub-complex, driving the assembly of the full complex.

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Section: Discussionsupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Merdes

Haren

Farache

et al. 2020

Preprint

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Proteolysis of γ‐tubulin small complex proteins is mediated by the ubiquitin‐proteasome system

et al. 2021

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Microtubule nucleation is mainly mediated by the γ‐tubulin ring complex (γTuRC), whose core components are γ‐tubulin and γ‐tubulin complex proteins GCP2–6. A substantial fraction of γ‐tubulin also exists with GCP2 and GCP3 in a tetramer called the γ‐tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ‐tubulin and GCPs have remained unclear. Here, we show that γ‐tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin‐proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ‐tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ‐tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components.

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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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Insights into the assembly and activation of the microtubule nucleator γ-TuRC

Cited by 126 publications

References 42 publications

A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Proteolysis of γ‐tubulin small complex proteins is mediated by the ubiquitin‐proteasome system

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

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