Reference: Riehlman TD, et al. (2013) Functional replacement of fission yeast ?-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3. J Cell Sci 126(Pt 19):4406-4413

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Abstract


UNLABELLED: Microtubule-organizing centers such as the ?-tubulin ring complex (?-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 ?-tubulin small complex (?-TuSC) of the ?-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 ?-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the ?-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 ?-TuSC proteins, we found that GCP3 assembles normally into the >2000 kDa fission yeast ?-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 ?-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast ?-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 ?-TuRC assembly. Our findings have broad importance for understanding the essential domains of ?-TuSC proteins in the ?-TuRC mechanism.

Reference Type
Journal Article
Authors
Riehlman TD, Olmsted ZT, Branca CN, Winnie AM, Seo L, Cruz LO, Paluh JL
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