INVITRO ASSEMBLY OF MULTIPROTEIN COMPLEXES CONTAINING ALPHA-TUBULIN, BETA-TUBLIN, AND GAMMA-TUBULIN, HEAT-SHOCK PROTEIN HSP70, AND ELONGATION FACTOR-1-ALPHA

被引:91
作者
MARCHESI, VT [1 ]
NGO, N [1 ]
机构
[1] YALE UNIV,SCH MED,DEPT PATHOL,NEW HAVEN,CT 06510
关键词
D O I
10.1073/pnas.90.7.3028
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 1alpha. 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.
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页码:3028 / 3032
页数:5
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