In vitro assembly of multiprotein complexes containing alpha, beta, and gamma tubulin, heat shock protein HSP70, and elongation factor 1 alpha.

Abstract

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 37C incubation in the presence of ATP or GTP, is composed of equivalent amounts of c-and f-tubulin and a 50-kDa protein, provisionally identified as elongation factor la. These complexes, which are heterogeneous in size when analyzed by sucrose gradient ultracentrifugation, also contain the cognate form of heat shock protein HSP70 and -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 37C 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.The genes and their protein products that initiate cell division and regulate its rate are being identified at a remarkable pace, in large part due to the realization that the mechanisms controlling cell growth are highly conserved across species (1). In contrast, our understanding of the proteins that make up the so-called mitotic apparatus, the organized complex of proteins that actually distributes the duplicated chromosomes to the daughter cells, has progressed much more slowly. To understand the complexity that underlies the assembly of mitotic spindles, it is necessary to learn how the extended microtubular cytoskeleton of an interphase cell becomes disassembled into its tubulin subunits at the appropriate point in the cell cycle and how these same tubulin units manage to reassemble into the two sets of polarized spindle tubules that become the functioning mitotic apparatus. Once nucleation of spindle tubules occurs, through processes that are now completely unknown, the growing tubules attach at one end to condensed chromosomes and at the other to a collection of proteins known as the centrosome or microtubule organizer center (MTOC). Centrosomes have been isolated and their capacity to nucleate microtubule assembly in vitro has been amply confirmed (2, 3), but the proteins responsible for this action have not been defined, nor has it been possible to disassemble isolated centrosomes into soluble, functional subunits. The results described below represent an attempt to isolate and characterize soluble protein precursors to centrosomes by preparing extracts of synchronized cells that are chemically blocked in a stage between prophase and metaphase. Reasoning that it might be easier to isola...

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

“…lndirect evidence indicates that EF-lu associates with MTs of the mitotic apparatus and that it may play a role in nucleating MTs (Ohta et al, 1988a(Ohta et al, , 1990Toriyama et al, 1988;Kuriyama et al, 1990;Marchesi and Ngo, 1993). However, a direct interaction of EF-lu with MTs has not been demonstrated; it is unclear whether previous investigators have attained the high leve1 of purity for EF-la preparations that we show here (Figure 3, lane c).…”

“…Binding between EF-1 as and Ca2+/CaM has not been described previously, and evidence for interactions between EF-1as and tubulin or MTs has been indirect (Ohta et al, 1988a(Ohta et al, , 1990Toriyama et al, 1988; Kuriyama et ai., 1990;Hasezawa and Nagata, 1993;Marchesi and Ngo, 1993). A direct binding between EF-la and MTs is demonstrated in this report.…”

Different functions of HOPS isoforms in the cell

“…Cytosolic complexes were isolated and fractionated by a 40-10% continuous sucrose gradient. 32 Immunoblot analysis revealed cosedimentation of HOPS with γ-tubulin, CRM-1, Ran-GAP, and NPM in the same fractions (Fig. 4B).…”

Moonlighting Functions of Polypeptide Elongation Factor 1: From Actin Bundling to Zinc Finger Protein R1-Associated Nuclear Localization

“…130) As EF-1a has in vitro a, b, and d tubulin binding activity, it has been suggested that EF-1a participates in the nucleation of astal microtubules, and in subsequent mitotic spindle formation. 154) Recently, we analyzed the localization of various EF-1 subunit-GFP fusion proteins in tobacco BY-2 cells. At the interphase, all the subunits tested (EF1a, b, and g) were colocalized with cytoplasmic F-actin.…”