ASSEMBLY PROPERTIES OF ALTERED BETA-TUBULIN POLYPEPTIDES CONTAINING DISRUPTED AUTOREGULATORY DOMAINS
ASSEMBLY PROPERTIES OF ALTERED BETA-TUBULIN POLYPEPTIDES CONTAINING DISRUPTED AUTOREGULATORY DOMAINS
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DOI：
10.1128/mcb.9.8.3418
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发表时间：
1989-08-01
影响因子：
5.3
通讯作者：
COWAN, NJ
中科院分区：
文献类型：
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作者：
GU, W;COWAN, NJ
β-真核细胞中的微管蛋白合成受转录后自动调节机制的控制，其中β-微管蛋白的前四个氨基酸被转录后自动调节机制调节。微管蛋白多肽直接或间接地控制β-微管蛋白的稳定性。微管蛋白mRNA。为了研究该氨基末端结构域对微管组装和动力学的贡献，我们引入了一系列缺失，包括单个哺乳动物β-微管的氨基酸2至5。微管蛋白同种型，M β 1。将携带这种缺失的构建体插入表达载体中，并通过使用抗M β 1特异性抗体测试改变的多肽共组装成微管的能力。我们表明，M β 1 β-当微管蛋白多肽缺少氨基酸2或氨基酸2和3时，其在瞬时转染实验中和稳定转染的细胞系中能够共组装成微管。这些突变体β的能力-微管蛋白共组装成聚合微管的能力相对于未改变的β-微管蛋白仅略微减少。微管蛋白，并且它们的表达不影响携带这些缺失的细胞系的生长特性的活力。然而，更广泛的氨基末端缺失严重损害或废除的能力，共组装。在类似的实验中，其中将改变引入哺乳动物α-氨基末端结构域，对于微管蛋白同种型M α 4，氨基酸2的缺失并不影响改变的多肽共组装的能力，尽管去除额外的氨基末端残基基本上消除了有效共组装的能力。改变的组装体-感受态α-和β-在脉冲追踪实验中测量微管蛋白多肽，发现其与相应的未改变的多肽的稳定性不可区分。携带包含12个羧基末端氨基酸的缺失的可组装M α 4多肽的半衰期也与野生型α 4多肽的半衰期不可区分。微管蛋白分子这些数据表明，β-氨基丁酸的普遍保守的氨基末端微管蛋白主要起调节作用，α-微管蛋白的羧基末端结构域微管蛋白不是哺乳动物细胞体内共组装所必需的。
.beta.-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the .beta.-tubulin polypeptide act either directly or indirectly to control the stability of .beta.-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian .beta.-tubulin isotype, M.beta.1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M.beta.1-specific antibody. We show that the M.beta.1 .beta.-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant .beta.-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered .beta.-tubulin, and their expression did not influence the viability of growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian .alpha.-tubulin isotype, M.alpha.4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent .alpha.- and .beta.-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M.alpha.4 polypeptide carrying a deletion encompassing the 12 carboxyterminal amino acids also had a half-life indistinguishable from that of the wild-type .alpha.-tubulin molecule. These data suggest that the universally conserved amino terminus of .beta.-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of .alpha.-tubulin is not essential for coassembly in mammalian cells in vivo.