Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3

Abstract

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

Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

“…To determine whether Cut7 binds the γ-TuRC MTOC by a similar mechanism, we performed Fast Protein Liquid Chromatography (FPLC)1517 using V5-tagged deletion derivatives of Cut7 previously generated29, immunocytology using newly generated V5-tagged deletion and site-directed mutagenesis derivatives, and Pkl1 peptide co-immunoprecipitation assays (Fig. 2).…”

“…All human γ-TuSC proteins are functional in fission yeast1415. We previously developed biochemical tools in the form of kinesin-14 Tail peptides (Fig.…”

“…12) and the γ-tubulin small complex (γ-TuSC) cryo-EM structure13. Conserved structural features are additionally supported by cross-species analysis1415. Still unknown is how dynamic control over MTOC functions for microtubule nucleation and organization is achieved.…”

“…Still unknown is how dynamic control over MTOC functions for microtubule nucleation and organization is achieved. The fission yeast Schizosaccharomyces pombe provides an ideal eukaryotic platform to address conserved MTOC mechanisms14151617.…”

“…Conservation of the kinesin-14 γ-TuRC regulatory mechanism is expected from yeast to human, as human kinesin-14 HSET replaces fission yeast kinesin-14 Pkl1 (ref. 23) and all human γ-TuSC protein components are also compatible1415. Nearly as ubiquitous and complex in eukaryotes as kinesin-14 Klps are members of the kinesin-5 family that oppose kinesin-14 function.…”