CHARACTERIZATION OF A MINUS END-DIRECTED KINESIN-LIKE MOTOR PROTEIN FROM CULTURED-MAMMALIAN-CELLS
CHARACTERIZATION OF A MINUS END-DIRECTED KINESIN-LIKE MOTOR PROTEIN FROM CULTURED-MAMMALIAN-CELLS
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DOI：
10.1083/jcb.129.4.1049
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发表时间：
1995-05-01
影响因子：
7.8
通讯作者：
KHODJAKOV, A
中科院分区：
文献类型：
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作者：
KURIYAMA, R;KOFRON, M;KHODJAKOV, A
使用针对 CHO 纺锤体产生的 CHO2 单克隆抗体（Sellitto, C.、M. Kimble 和 R. Kuriyama. 1992. Cell Motil. Cytosculpt. 22:7-24），我们鉴定了位于间期中心体和有丝分裂纺锤体处的 66-kD 蛋白质。分离的抗原 cDNA 编码 622 个氨基酸的多肽。序列分析揭示了 COOH 末端存在 340 个氨基酸残基，这与驱动蛋白超家族其他成员中保守的运动结构域同源。该蛋白质由中央 α 螺旋部分组成，在 NH2 和 COOH 末端均具有球状结构域，单克隆抗体的表位位于中央 α 螺旋柄中。创建了一系列缺失构建体用于微管相互作用的体外分析。虽然微管结合和成束活性需要 COOH 末端和 α 螺旋结构域的存在，但抗原的 NH2 末端一半缺乏与微管相互作用的能力。由COOH末端马达和中央α螺旋柄组成的全长蛋白质和缺失蛋白质支持微管滑动，速度范围为1.0至8.4μm/分钟。微管运动的速度随着连接到 COOH 末端马达的中央柄的长度的减小而降低。微管以正端引导移动，表明抗原是负端引导的运动。 CHO2 序列与 HSET 具有 86% 的同一性，HSET 是位于 6 号染色体中人类 MHC 区域着丝粒末端的基因（Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. 免疫遗传学。 39:194-200)，表明 HSET 可能代表 CHO2 抗原的人类同源物。
Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 mu m/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.