Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function
Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function
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DOI：
10.1074/jbc.m604817200
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发表时间：
2006-09-08
影响因子：
4.8
通讯作者：
Hoyt, M. Andrew
中科院分区：
文献类型：
--
作者：
Hildebrandt, Emily R.;Gheber, Larisa;Hoyt, M. Andrew
驱动蛋白-5马达蛋白在进化上是保守的，并且在有丝分裂后期的纺锤体组装和纺锤体延伸中发挥重要作用。以前的研究表明，一个专门的同源四聚体结构与两对催化结构域，一个在哑铃形分子的两端。这表明它们通过交联和滑动反平行纺锤体微管来执行纺锤体的作用。然而，确切的驱动蛋白-5序列元素，是重要的四聚体复合物的形成尚未确定。此外，尚未证明这些蛋白质的同源四聚体形式对其生物学功能是必需的。因此，我们研究了一系列酿酒酵母Cin 8 p截短和内部缺失，以确定Cin 8 p序列中Cin 8 p功能，纺锤体定位和多聚化所需的结构元件。我们发现在体内有功能的Cin 8 p的所有变体形成四聚体复合物。Cin 8 p茎中的第一卷曲螺旋结构域是其二聚化所需的，这是所有驱动蛋白-5同源物共有的特征，并且茎的最后部分中的序列，特别是那些可能参与卷曲螺旋形成的序列，是Cin 8 p四聚化所需的。我们还发现，体内无功能的Cin 8 p的二聚体形式仍然可以与微管结合。这些发现表明微管的结合对于Cin 8 p的功能是不够的，并且通过四聚体复合物的微管交联对于Cin 8 p有丝分裂功能是必不可少的。
Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.