Tubulin Subunits Exist in an Activated Conformational State Generated and Maintained by Protein Cofactors

Abstract

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

Tubulin Folding Cofactors as GTPase-activating Proteins

“…Cofactors as ␤-Tubulin GTPase-activating Proteins-We have previously shown that when tubulin and either cofactors C and D or cofactors C, D and E are mixed, GTP hydrolysis ensues (11). To understand the nature of this reaction, we measured the rate of GTP hydrolysis as a function of the concentration of added tubulin ( Fig.…”

“…Here we show that both in the folding reaction and in the related reaction of cofactors with native tubulin, the cofactors act as GTPase-activating proteins (GAPs), 1 stimulating many-fold the negligible intrinsic GTPase activity of the tubulin to which they are bound. 35 S-labeled in the ␣-or ␤-subunit, full-length tubulin cDNAs encoding mouse ␣2 (13) or mouse ␤3 (14) were 35 S-labeled by coupled transcription/translation in rabbit reticulocyte lysate (Promega Inc., Madison, WI) and purified by anion exchange chromatography on DEAE-Sephacel (11). Tubulin folding cofactors A, B, C, and D were either purified from bovine testis tissue or from Escherichia coli (as cloned recombinant proteins) as described (9 -11).…”

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

“…Whereas fully functional actin and ␥-tubulin polypeptides are released from the cytosolic chaperonin complex, ␣-tubulin and ␤-tubulin polypeptides are further processed by tubulin-folding cofactors (TFCs; for review, see Lewis et al 1997;Nogales 2000). TFCs were originally identified by their tubulin-folding activity in extracts from mammalian cells (Gao et al 1994;Llosa et al 1996;Melki et al 1996;Tian et al 1996Tian et al , 1997. In vitro folding assays suggest that ␣-tubulin binds to TFCs B and E, whereas ␤-tubulin binds to TFCs A and D .…”

Revisiting the tubulin folding pathway: new roles in centrosomes and cilia

“…Nevertheless, TBCA is an essential gene in human cell lines with a preponderant role in the recycling of mature tubulin heterodimers (54) (Nolasco et al, unpublished). TBCB, TBCE and TBCD have the ability of dissociating the tubulin dimer, causing microtubule depolymerization when overexpressed in mammalian cells (55)(56)(57)(58). TBCB overexpression also leads to a decrease in the number of microtubules in plant cells (59).…”