Functionally distinct isoforms of dynactin are expressed in human neurons
Functionally distinct isoforms of dynactin are expressed in human neurons
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DOI：
10.1091/mbc.7.8.1167
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发表时间：
1996-08-01
影响因子：
3.3
通讯作者：
Holzbaur, ELF
中科院分区：
文献类型：
--
作者：
Tokito, MK;Howland, DS;Holzbaur, ELF
p150(Glued) 是 dynactin 的最大亚基，它与细胞质动力蛋白结合并激活囊泡沿微管的运输。我们分离出了编码 p150(Glued) 以及 135-kDa 亚型的人类 cDNA；这些亚型通过人 DCTN1 基因的选择性 mRNA 剪接在人脑中表达。 p135 亚型缺乏 p150(Glued)/Glued/CLIP-170/BIK1 微管相关蛋白家族成员共有的共有微管结合基序，因此预计不会直接与微管结合。我们使用瞬时转染测定和体外微管结合测定来证明 p150 亚型与微管结合，但 p135 亚型则不结合。然而，两种亚型均与细胞质动力蛋白结合，并且都类似地分配为细胞质和膜细胞部分。使用 p150 异构体特异性抗体和随后的抗体进行的顺序免疫沉淀显示，在大脑中，这些多肽组装形成不同的复合物，每个复合物在类似于 20 S 的温度下沉积。根据这些观察结果，我们假设不同形式的 dynactin 复合物具有保守的神经元功能，不能直接与细胞微条结合。
p150(Glued) is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150(Glued) as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150(Glued)/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at similar to 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtibules.