Activation of the γ-Tubulin Complex by the Mto1/2 Complex

Abstract

SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.

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

“…Thus, heterogeneity could explain the poor quality of the NMR spectra, which is likely due to chemical exchange between the different states of MOZART1 in the sample leading to NMR line broadening. The presence of MOZART1 oligomers in solution may reflect a role of this protein in the lateral assembly of multiple γTuSCs into γTuRCs, similar to the previously documented role of Spc110 in γTuRC assembly …”

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

“…Among them, Cnn-related/CM1-containing proteins, including Drosophila Cnn, mammalian CDK5RAP2 and Myomegalin, fission yeast Mto1 and Pcp1, etc., appear to be the most potent stimulators for MT nucleation by γ-TuRCs, as shown by in vitro biochemical assays and/or by redirecting the protein (or protein fragments) to ectopic cellular locations to induce MT assembly at those sites (this study, and [20, 28, 32, 45]). Our data further support that Cnn (and orthologs) directly binds and stimulates γ-TuRCs to mediate MT assembly.…”

“…In S. pombe, a shortened “bonsai” version of the Cnn homolog Mto1p, that contains the CM1 domain and a binding site for Mto2p, allows for binding of γ-TuCs and oligomerization, enabling it to activate the γ-TuC without associating with an MTOC [45]. CM1 of human CDK5RAP2, however, is sufficient for γ-TuRC recruitment and MTOC activity [20].…”

MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation

“…In yeast, activation might result from γTuSC oligomerization (Kollman et al, 2010;Lin et al, 2014;Lynch et al, 2014). In human cells, however, oligomerization per se might not be sufficient for full nucleation activity: even though oligomeric complexes exist in the form of pre-assembled γTuRCs, the 50-amino-acid CM1 motif of human CDK5RAP2 alone is able to strongly stimulate nucleation activity of these complexes in vitro and in vivo (Choi et al, 2010).…”

“…As a result, γTuSC oligomerizes and becomes a more active nucleation template (Lin et al, 2014). Even in fission yeast, which contains GCP4, GCP5 and GCP6, formation of active higherorder γ-tubulin complexes requires interaction with the CM1-containing protein Mto1 that forms an adapter complex with Mto2 at interphase MTOCs (Lynch et al, 2014). Adapter-mediated γTuSC oligomerization at MTOCs might also explain how the experimental loss of the γTuRC-specific subunits GCP4, GCP5 and GCP6 is compensated for in flies (Vérollet et al, 2006).…”