MULTIPLE FORMS OF TUBULIN IN THE CYTOSKELETAL AND FLAGELLAR MICROTUBULES OF POLYTOMELLA
MULTIPLE FORMS OF TUBULIN IN THE CYTOSKELETAL AND FLAGELLAR MICROTUBULES OF POLYTOMELLA
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DOI：
10.1083/jcb.91.2.352
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发表时间：
1981-01-01
影响因子：
7.8
通讯作者：
ROSENBAUM, JL
中科院分区：
文献类型：
--
作者：
MCKEITHAN, TW;ROSENBAUM, JL
多壳藻含有多个由微管组成的细胞器，包括4个鞭毛和数百个细胞骨架微管。Brown和他的同事（1976年）指出，鞭毛可以被去除，细胞骨架可以被分离，并且在缺乏蛋白质合成的情况下，这两种结构都可以部分再生。正因为如此，因为鞭毛和细胞骨架可以被完整地分离出来，这种生物体特别适合于研究微管蛋白的异质性和特定的微管蛋白在同一细胞中掺入不同的微管细胞器。为了确定在Polytomella细胞质中的微管蛋白的不同种类，在未标记的脑微管蛋白的存在下，使35 S标记的细胞质级分经历2个循环的组装和拆卸。通过二维凝胶电泳比较通过该方法获得的标记的多囊藻胞质微管蛋白与分离的多囊藻鞭毛的微管蛋白显示，而β-微管蛋白的表达与分离的多囊藻鞭毛的微管蛋白的表达不同。来自细胞质和鞭毛微管蛋白样品的微管蛋白共迁移，2 α-微管蛋白具有明显不同的等电点。作为从细胞质中分离微管蛋白的第二种方法，用去污剂轻轻裂解细胞并获得完整的细胞骨架。当这些细胞骨架暴露在低温下时，释放的蛋白质高度富集在微管蛋白中;这种微管蛋白本身可以在体外组装成微管。主要的α-这种体外组装的细胞骨架微管蛋白的微管蛋白对应于主要的细胞质α-微管蛋白。微管蛋白通过标记的Polytomella胞质提取物与脑微管蛋白的共组装获得，并且与α-微管蛋白非常不同。纯化的鞭毛微管蛋白。来自同一细胞的两种不同的含微管的细胞器由不同的微管蛋白组成。
The alga Polytomella contains several organelles composed of microtubules, including 4 flagella and hundreds of cytoskeletal microtubules. Brown and co-workers showed (1976) that the flagella could be removed and the cytoskeletons dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in Polytomella cytoplasm, a 35S-labeled cytoplasmic fraction was subjected to 2 cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled Polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated Polytomella flagella by 2 dimensional gel electrophoresis showed that, whereas the .beta.-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the 2 .alpha.-tubulins had distinctly different isoelectric points. As a 2nd method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant .alpha.-tubulin of this in vitro-assembled cytoskeletal tubulin corresponded to the major cytoplasmic .alpha.-tubulin obtained by coassembly of labeled Polytomella cytoplasmic extract with brain tubulin and was quite distinct from the .alpha.-tubulin of purified flagella. Two different microtubule-containing organelles from the same cell are composed of distinct tubulins.