title: Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity.
abstract: Assembly of the α/β tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and β-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with β-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with β-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by β-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein).
authors.affiliation: Department of Biochemistry, NYU Langone Medical Center, 550 First Avenue, New York, New York 10016, USA.
authors.forename: Guoling
authors.initials: G
authors.lastname: Tian
authors.forename: Simi
authors.initials: S
authors.lastname: Thomas
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Cowan
mesh_terms: D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008869:Microtubule-Associated Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C418662:TBCD protein, human; D014404:Tubulin; C410130:ARL2 protein, human; D019204:GTP-Binding Proteins