In vitro assembly of multiprotein complexes containing alpha, beta, and gamma tubulin, heat shock protein HSP70, and elongation factor 1 alpha.
We have isolated two sets of multiprotein complexes from supernatants from high-speed centrifugation of nocodazole-arrested CHO cells. One set, assembled in vitro after a 37 degrees C incubation in the presence of ATP or GTP, is composed of equivalent amounts of alpha- and beta-tubulin and a 50-kDa protein, provisionally identified as elongation factor 1 alpha. These complexes, which are heterogeneous in size when analyzed by sucrose gradient ultracentrifugation, also contain the cognate form of heat shock protein HSP70 and gamma-tubulin, a tubulin isoform of low abundance, along with other proteins known to be involved in the regulation of mitosis. Similar but distinct complexes assemble in vitro if the same extracts are incubated at 37 degrees C without added nucleotides; multiprotein complexes generated under these conditions lack HSP70 but contain instead a 43-kDa protein identified as an actin isoform. Both sets of assembled complexes exhibit a globular substructure when analyzed by electron microscopy, and their size distribution suggests that they assemble by the step-wise addition of smaller precursors. The properties of these multiprotein complexes and their presence in cells arrested in a stage between prophase and metaphase suggest that they may be precursors to mitotic centrosomes and are possibly involved in mitotic spindle nucleation.
Marchesi
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Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT 06536-0812.
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D000818:Animals; D016466:CHO Cells; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008938:Mitosis; D008961:Models, Structural; D008970:Molecular Weight; D015739:Nocodazole / Q000494:pharmacology; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000032:analysis / Q000378:metabolism; D014404:Tubulin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012261:Ribonucleoproteins; D014404:Tubulin; D015739:Nocodazole