The Kar3p kinesin-related protein forms a novel heterodimeric structure with its associated protein Cik1p
The Kar3p kinesin-related protein forms a novel heterodimeric structure with its associated protein Cik1p
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DOI：
10.1091/mbc.11.7.2373
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发表时间：
2000-07-01
影响因子：
3.3
通讯作者：
Snyder, M
中科院分区：
文献类型：
--
作者：
Barrett, JG;Manning, BD;Snyder, M
Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins.然而，驱动蛋白和驱动蛋白相关蛋白之间的复合物的四级结构尚不清楚。我们分析了 Kar3 运动蛋白（一种来自酵母的负端定向驱动蛋白）与其相关蛋白 Cik1 之间相互作用的性质。 Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated.通过双杂交分析对两种蛋白质的相互作用结构域进行绘图表明，Kar3p 和 Cik1p 沿着各自卷曲螺旋结构域的长度以高度特异性的方式关联。使用蔗糖梯度速度离心和凝胶过滤实验来测定交配信息素处理的细胞和营养生长细胞的 Kar3-Cik1 复合物的大小。这些实验预测该复合物的大小与包含一个 Kar3p 亚基和一个 Cik1p 亚基的异二聚体的大小一致。最后，表位标记和未标记蛋白质的免疫沉淀证实了 Kar3-Cik1 复合物中仅存在 Kar3p 和 Cik1p 的一个亚基。这些发现表明，Kar3-Cik1 复合物具有以前在驱动蛋白复合物中未观察到的新型异二聚体结构。
Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.