Expression of a human .alpha.-tubulin: properties of the isolated subunit

Yaffe, Michael B.; Levison, Bruce S.; Szasz, J.; Sternlicht, H.

doi:10.1021/bi00406a012

Cited by 31 publications

(30 citation statements)

References 76 publications

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“…Therefore, it seems reasonable that de novo synthesized a-tubulin might exchange within the heterodimer pool, and thus become dimer (mouse a-rabbit P). Because in vitro synthesized a-tubulin is indistinguishable in size, charge and polymerization properties from dimers [Yaffe et al, 1988b; and this work], we believe that this species represents tubulin dimer.…”

Section: Discussionmentioning

confidence: 70%

“…Also, we could not detect the presence of a-tubulin monomers even at early times after a kinetic analysis of de novo synthesis of different a-tubulin isotypes. Yaffe et al [1988b] suggested that the in vitro synthesis of a-tubulin generates monomeric subunits but not dimeric forms. This conclusion was based on the observation that the a-tubulin product could not be immunoprecipitated with a P-tubulin monoclonal antibody and that its protease digestion pattern differed from that of cycled, translated a-tubulin.…”

Section: Discussionmentioning

confidence: 99%

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Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes

Zabala

Cowan

1992

Cell Motil. Cytoskeleton

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The functional subunit of microtubules is a heterodimer consisting of alpha- and beta-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse alpha-tubulin isotypes involves a multimolecular complex. The synthesis of mouse beta-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized alpha- and beta-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers.

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Section: Discussionmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes

Zabala

Cowan

1992

Cell Motil. Cytoskeleton

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“…1C, lanes 7 and 12). In contrast, the translation of mRNAs encoding either a-or ,B-tubulin in unfractionated rabbit reticulocyte lysate leads to the synthesis of assembly-competent tubulin (6,51). These data led us to suspect that some factor or factors in addition to chaperonin itself must be required for the proper folding of a-and 13-tubulin.…”

Section: Resultsmentioning

confidence: 99%

Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin.

et al. 1993

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Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood. We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y. Gao, J. 0. Thomas, R. L. Chow, G.-H. Lee, and N. J. Cowan, Cell 69:1043-1050, 1992). Here we show that the same chaperonin also forms a binary complex with unfolded a-or ,-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded a-or 13-tubulin polypeptides might exchange. Rather, two additional protein cofactors are required for the generation of properly folded a-or 13-tubulin, which is then competent for exchange into preexisting al-tubulin heterodimers. We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes. These data imply that actin and a-and 13-tubulin interact with the same site(s) on chaperonin complexes.There is compelling evidence that all of the information required for a given protein to assume its correct threedimensional structure is contained within its primary sequence (2, 24). Though this implies that the folding of proteins in vivo can occur spontaneously, it is now clear that in many cases folding is facilitated via interaction with one or more members of a class of proteins known as molecular chaperones or polypeptide chain-binding proteins (7,36). The majority of currently identified chaperones belong to one of two conserved families, exemplified by the stressinduced (though also constitutively expressed) heat shock proteins hsp70 and hsp60; the latter are also termed chaperonins (10,15,35). Both groups use the energy of ATP hydrolysis to release their bound substrate proteins and seem to act by stabilizing the conformation of folding intermediates, thereby preventing the formation of aberrant structures and directing the polypeptides toward correct folding, assembly, and translocation (5,16,17,21,25,38,46). Although hsp70 proteins and chaperonins share some common functional features, their roles are not interchangeable, and they are structurally distinct: while members of the hsp7O family act as monomers or dimers, chaperonins are multisubunit toroidal complexes (3-5, 8, 14, 16, 17, 22, 23, 33, 34, 44).The mechanisms involved in chaperonin-mediated protein folding are not well understood. In the case of the bacterial chaperonin (GroEL), there is evidence that the chaperonin can recognize secondary structure elements (28), which could result in the stabilization of conformational intermediates bound to the chaperonin (32). The folding reaction requires the hydrolysis of about 100 mol of ATP per mol of protein folded and may involve the ordered, st...

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“…It should be pointed out that while the idea that et-tubulin represses its own synthesis is consistent with the data, we cannot presently rule out the possibility that 13-tubulin, alone or in combination with some other factor, binds to et-tubulin m R N A and directly promotes translation. However, there is currently little evidence for this type of translational control, except in viral systems (Standart and Jackson, 1994), and the fact that et-tubulin can be successfully translated in vitro in the absence of new 13-tubulin synthesis (Yaffe et al, 1988) leads us to prefer a mechanism in which et-tubulin acts as a repressor of its own translation. Whatever the actual mechanism, our results suggest that a-tubulin synthesis is controlled by the availability of [3-tubulin for heterodimer formation.…”

Section: Discussionmentioning

confidence: 99%

alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression.

Gonzalez-Garay

Cabral

1996

The Journal of Cell Biology

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Abstract.A Chinese hamster a-tubulin cDNA was modified to encode an ll-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HAaltubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HAal-tubulin were isolated and characterized. HAal-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HAed-tubulin strongly repressed the synthesis of endogenous a-tubulin while having little or no effect on the synthesis of [3-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HAal-tubulin led to a further decrease in synthesis of endogenous ot-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of et-tubulin in transfected cells did not result from decreased levels of a-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HAaltubulin and endogenous a-tubulin, thus indicating that the decreased et-tubulin synthesis observed as a result of HAed-tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free et-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of a-and 13-tubulin.

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Expression of a human .alpha.-tubulin: properties of the isolated subunit

Cited by 31 publications

References 76 publications

Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes

Tubulin dimer formation via the release of α‐ and β‐tubulin monomers from multimolecular complexes

Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin.

alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression.

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