title: Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3.
abstract: Microtubule-organizing centers such as the γ-tubulin ring complex (γ-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast γ-tubulin small complex (γ-TuSC) of the γ-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast γ-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the γ-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human γ-TuSC proteins, we found that GCP3 assembles normally into the >2000 kDa fission yeast γ-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of γ-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast γ-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during γ-TuRC assembly. Our findings have broad importance for understanding the essential domains of γ-TuSC proteins in the γ-TuRC mechanism.
authors.affiliation: College of Nanoscale Science and Engineering, State University of New York (SUNY), Nanobioscience, Albany, NY12203, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Riehlman
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Olmsted
authors.forename: Carmen N
authors.initials: CN
authors.lastname: Branca
authors.forename: Adam M
authors.initials: AM
authors.lastname: Winnie
authors.forename: Lan
authors.initials: L
authors.lastname: Seo
authors.forename: Leilani O
authors.initials: LO
authors.lastname: Cruz
authors.forename: Janet L
authors.initials: JL
authors.lastname: Paluh
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C512340:Alp4 protein, S pombe; C512341:Alp6 protein, S pombe; D008869:Microtubule-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; C112305:TUBGCP2 protein, human; C112306:TUBGCP3 protein, human; D014404:Tubulin
keywords: MTOC; Spc97; Spc98; γ-TuRC; γ-TuSC