BETA-CENTRACTIN - CHARACTERIZATION AND DISTRIBUTION OF A NEW MEMBER OF THE CENTRACTIN FAMILY OF ACTIN-RELATED PROTEINS
BETA-CENTRACTIN - CHARACTERIZATION AND DISTRIBUTION OF A NEW MEMBER OF THE CENTRACTIN FAMILY OF ACTIN-RELATED PROTEINS
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DOI：
10.1091/mbc.5.12.1301
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发表时间：
1994-12-01
影响因子：
3.3
通讯作者：
MEYER, DI
中科院分区：
文献类型：
--
作者：
CLARK, SW;STAUB, O;MEYER, DI
对人类表达的序列标签的检查表明，存在一种异构体的肌动蛋白，一种定位于微管相关结构的肌动蛋白相关蛋白。利用其中一个标签，我们分离并测定了一个全长cDNA克隆的核苷酸序列。编码的蛋白质代表了单个生物体中肌动蛋白相关蛋白的多个异构体的第一个例子。Northern分析发现HeLa细胞中有三种可以编码中心肌动蛋白异构体的mRNA。一个信使核糖核酸编码以前发现的中央肌动蛋白(现在被称为α-中央肌动蛋白)。利用表达序列标签分离到的全长cDNA克隆编码一个新成员--β-Centactin。一种与所观察到的第三种信使核糖核酸(称为伽马中央肌动蛋白)杂交的针对α-中央肌动蛋白的探针。对人体组织Northern blotts的比较表明，α-中心肌动蛋白和β-中心肌动蛋白mRNAs在所有被研究的mRNA群体中均匀分布，而γ-中心肌动蛋白的表达似乎是组织特有的。由该基因推导出的β-中央肌动蛋白的氨基酸序列与α-中央肌动蛋白有91%的同源性，如果考虑到保守的氨基酸变化，则相似性增加到96%。由于先前提出的针对α-中央肌动蛋白的抗体与β-中央肌动蛋白反应很差，因此产生了新的抗体，并与双向凝胶电泳法相结合来区分这两种异构体。利用这个系统，我们确定了α-和β-异构体的亚细胞分布。这两种异构体主要存在于胞浆部分，是以前发现的20S复合体(称为动力蛋白复合体)的一部分，没有证据表明这两种异构体都存在自由池。在动力蛋白复合体中，异构体的比例接近15：1(α：β)。
An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of similar to 15:1 (alpha:beta) in the dynactin complex.