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. 1997 Aug 25;138(4):821-32.
doi: 10.1083/jcb.138.4.821.

Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors

G Tian  1 , S A Lewis, B Feierbach, T Stearns, H Rommelaere, C Ampe, N J Cowan
Affiliations

Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors

G Tian (VSports最新版本) et al. J Cell Biol. .

Abstract

The production of native alpha/beta 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 beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-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 alpha-tubulin folding in vitro VSports手机版. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-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 alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other. .

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Figure 1
Figure 1
Characterization of cofactor B. (A) The cofactors that support productive β-tubulin folding do not efficiently support α-tubulin folding. Analysis by nondenaturing gel electrophoresis of the products of c-cpn–mediated β-tubulin (lane 1) and α-tubulin (lane 2) folding reactions done with cofactors A, C, D, and E. (B) A novel cofactor (cofactor B) is involved in α-tubulin folding. Analysis of the products of c-cpn–mediated α-tubulin folding reactions done in the presence of aliquots of fractions obtained by anion exchange chromatography of an extract of bovine testis tissue. Only assays of those fractions emerging from the column in the range of 50–140 mM MgCl2 are shown. (C and D) Purification of cofactor B. Absorbance profile of the final step (gel filtration) in the purification of cofactor B (C), together with SDS-PAGE of material contained in the major peak emerging from the column (D). In D, the location of molecular size markers (in kD) is shown. (E and F) The fast-migrating species produced in c-cpn– mediated α-tubulin folding reactions containing purified cofactor B does not cocycle efficiently with native brain tubulin. The products of a c-cpn–mediated α-tubulin folding reaction done in the presence of purified cofactor B alone (E) or cofactors A, B, C, D, and E (F) were subjected to successive cycles of polymerization and depolymerization with added native bovine brain tubulin. The specific activity (relative to the material obtained after the first cycle, taken as 100%) after each cycle is shown. (A and B, upper and lower arrows). Location of α- or β-tubulin/c-cpn binary complexes and either native tubulin (A) or the product generated by bovine cofactor B (B).
Figure 3
Figure 3
Benomyl sensitivity of alf1, cin1, and pac2 mutants. Suspensions of cells were spotted onto yeast extract/peptone/dextrose plates containing either no benomyl (as a control) or benomyl at 5 or 10 μg/ml and grown at 30°C. The strains assayed had null mutations in the genes shown in the figure and were of the same genetic background.
Figure 4
Figure 4
Cofactors that participate in α-tubulin folding in vitro. (A) Bovine and recombinant human cofactor B form complexes with c-cpn–generated α-tubulin folding intermediates that have different mobilities on a nondenaturing gel. Products of c-cpn– mediated α-tubulin folding reactions done without cofactors (lane 1) or with added purified bovine (lane 2) or recombinant human (lane 3) cofactor B are shown. (B and C) Cofactors required for productive α-tubulin folding in vitro. C-cpn–mediated α-tubulin folding reactions were done in the presence of native tubulin and the cofactors shown in the figure. (D) Native tubulin can be distinguished from the FBα complex by digestion with subtilisin. Analysis of native bovine brain tubulin detected by staining with Coomassie blue (lanes 1 and 2), or the products of in vitro c-cpn–mediated α-tubulin folding reactions containing either cofactor B alone (lanes 3 and 4) or cofactors B, C, D, and E (lanes 5 and 6). Reaction products were either analyzed directly (lanes marked −) or after digestion with subtilisin (lanes marked +). (E) The α-tubulin target protein in FBα contains nonexchangeably bound GTP and can be partitioned to the native state by the action of cofactors C, D, and E, and native tubulin. Analysis of the products of c-cpn–mediated α-tubulin folding reactions done with unlabeled target protein in the presence of α-[32P]GTP and recombinant human cofactor B alone either directly (lane 1), after isolation of the FBα intermediate by gel filtration (lane 2), or after incubation of the latter material in the presence of cofactors C, D, and E (lane 3); all reactions contained added native tubulin. (F) Unstable intermediates formed by reaction of FBα (generated using bovine cofactor B and containing bound 32P-labeled GTP) with cofactors D and E. Nondenaturing gel electrophoresis of reaction products after incubation of isolated FBα with the components shown in the figure. These reaction products were stabilized by brief treatment with glutaraldehyde before application to the gel (see text and Materials and Methods). Note that FBα formed with bovine cofactor B comigrates with native tubulin dimer. (C and F, asterisk). A band of intermediate mobility that appears in reactions containing native tubulin and cofactors D and E (but not C) (see text). (Arrows, top to bottom): the location of α-tubulin/c-cpn binary complexes, the human FBα complex, and either the bovine FBα complex or native tubulin, respectively.
Figure 5
Figure 5
Interaction of cofactors with native tubulin subunits. (A) Intermediates produced via reaction of cofactors with native tubulin dimers. Purified native tubulin 35S-labeled in either the α or β subunit was incubated for 1 h at 30°C at a concentration of 0.05 μM with either added unlabeled purified native tubulin (2.5 μM) or with a fivefold molar excess of the cofactors shown in the figure. (B) Purified native tubulin 35S-labeled in the β subunit was incubated for 1 h at 30°C at a concentration of 2.5 μM either without or with a 1.5-fold molar excess of cofactor D. (C) Free α-tubulin subunits generated by sequestration of β-tubulin by cofactor D can be trapped by mitochondrial chaperonin (mt-cpn). Purified native tubulin 35S-labeled in the α subunit was first incubated at 30°C with a 1.5-fold molar excess of cofactor D so as to sequester the β subunits. At t = 0 or t = 15 min thereafter, 2.5 μM purified tubulin, a fivefold molar excess of cofactor B or E, or a 50-fold molar excess of mt-cpn was added, and the incubation continued for an additional 30 min. As a control, native tubulin 35S-labeled in the α subunit was incubated alone with the same molar excess of mt-cpn. (D) Free β-tubulin subunits generated by sequestration of α-tubulin by cofactors B and E are also capturable by an mt-cpn trap. Purified tubulin 35S-labeled in the β subunit was incubated at 30°C for 1 h in the presence of unlabeled tubulin, or a 10-fold molar excess of cofactors B and E without or with a 50-fold molar excess of trap. As a control for the effect of trap on native tubulin itself, the input labeled tubulin was incubated in a parallel reaction with the same molar excess of trap but without addition of cofactors. (A–D) Reaction products were analyzed on 4.5% nondenaturing polyacrylamide gels. Upper and lower arrows on the left denote the location of the human FBα complex and native tubulin heterodimer, respectively; arrows on the right denote the location of mt-cpn. (A and C) Bands produced in reactions done with cofactors D and E are highlighted with an asterisk (see text).
Figure 6
Figure 6
Role of GTP hydrolysis in tubulin folding. (A) Tubulin folding is accompanied by GTP hydrolysis. In vitro c-cpn–mediated α- or β-tubulin folding reactions were done in the presence of cofactors without or with GTPγS (see Materials and Methods). (B) GTPγS, but not ATPγS, blocks the release of both α- and β-tubulin from complexes containing cofactors D and E. Purified native tubulin heterodimers 35S-labeled in either the α or β subunit were incubated at 30°C for 1 h in the presence of 10 μM GTP with a 1.5-fold molar excess of cofactors D or D plus E to generate tubulin/cofactor complexes (see Fig. 4 A). To complete the reaction, cofactor C and unlabeled tubulin (2.5 μM) were added, and the incubation continued in the presence or absence of GTPγS or ATPγS for an additional hour. (A and B) Reaction products were analyzed on 4.5% nondenaturing polyacrylamide gels. Upper and lower arrows show the location of c-cpn/tubulin binary complex and native tubulin heterodimer, respectively. (C) GTP hydrolysis by cofactors and by tubulin/cofactor complexes. The percentage conversion to GDP is shown. (D) GTP can be cross-linked to β-tubulin in the α/β-supercomplex. Analysis on an 8% SDS–polyacrylamide gel of the UV cross-linked products of a reverse tubulin folding reaction containing cofactors C, D, E, and α–32P-labeled GTP without (lane 1) or with native tubulin heterodimer (lane 2). The location of molecular mass markers (in kD) is shown, together with the location of α- and β-tubulin.
Figure 7
Figure 7
Convergence and symmetry of the α- and β-tubulin folding pathways (see text). Quasinative α- and β-tubulin folding intermediates produced via ATP-dependent interaction with c-cpn (shown as eight-subunit toroids) interact with a series of protein cofactors (FA, FB, FC, FD, and FE). The pathways converge via the formation of a complex containing α- and β-tubulin and cofactors D and E (asterisk). Entry of cofactor C generates the α/β-supercomplex (boxed); GTP hydrolysis then results in the release of native polypeptides. Broken arrows show the backreaction between native α- or β-tubulin subunits and cofactors E or D. The names of tubulin/cofactor complexes are not intended to reflect their stoichiometry. α, α-tubulin target protein. β, β-tubulin target protein.
Figure 2
Figure 2
Amino acid sequence of cofactor B: homology with cofactor E and the S. cerevisiae protein Alf1p. Amino acid sequences of peptides derived from purified bovine cofactor B, together with the deduced amino acid sequences of human cofactor B (these sequence data are available from EMBL/GenBank/ DDBJ under accession number AF013488), the S. cerevisiae homologue Alf1p, and the microtubule binding motif in cofactor E. The corresponding repeated motifs in CLIP-170 are shown for comparison.
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