title: Human SAS-6 C-Terminus Nucleates and Promotes Microtubule Assembly in Vitro by Binding to Microtubules.
abstract: Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Hindol
authors.initials: H
authors.lastname: Gupta
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Binshad
authors.initials: B
authors.lastname: Badarudeen
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Athira
authors.initials: A
authors.lastname: George
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Geethu Emily
authors.initials: GE
authors.lastname: Thomas
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: K K
authors.initials: KK
authors.lastname: Gireesh
authors.affiliation: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram , CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Manna
mesh_terms: D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D014404:Tubulin / Q000032:analysis / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C497202:SASS6 protein, human; D014404:Tubulin