Activation of the γ-tubulin complex by the Mto1/2 complex.
The multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells, but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm. 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. 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.
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK.
Eric M
EM
Lynch
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK.
Lynda M
LM
Groocock
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK.
Weronika E
WE
Borek
Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK. Electronic address: ken.sawin@ed.ac.uk.
Kenneth E
KE
Sawin
D002352:Carrier Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000502:physiology* / Q000648:ultrastructure; D046912:Multiprotein Complexes / Q000378:metabolism* / Q000648:ultrastructure; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D059008:Time-Lapse Imaging; D014404:Tubulin / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; D000097573:Red Fluorescent Protein; C538717:Mto1 protein, S pombe; C505279:mto2p protein, S pombe