title: Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.
abstract: Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.
authors.affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Kollman
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Polka
authors.forename: Alex
authors.initials: A
authors.lastname: Zelter
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
authors.forename: David A
authors.initials: DA
authors.lastname: Agard
mesh_terms: D002021:Buffers; D002148:Calmodulin-Binding Proteins; D020285:Cryoelectron Microscopy; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D002021:Buffers; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; C465662:SPC97 protein, S cerevisiae; C552429:SPC98 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin