Analysis of the functions of a microtubule-interacting protein, YTM1, which is essential for the G1/S transition and of its related genes.

微管在细胞运动、运输、细胞形状和极性以及有丝分裂等许多细胞过程中发挥重要作用。阐明控制微管组织的机制具有重要意义。我们已经分离出YTM 1作为酵母β-微管蛋白突变体的多拷贝抑制因子，其中单个β-微管蛋白的羧基末端可变结构域被删除。YTM 1编码一个460个氨基酸的新蛋白，具有β-转导素（WD-40）重复序列。间接免疫荧光法显示YTM 1主要集中在细胞核内，并与纺锤体微管共定位。与聚合猪脑微管蛋白的共沉淀实验表明YTM 1可以与微管结合。YTM 1是细胞生长所必需的，其功能缺陷导致细胞周期停滞在G1期DNA含量较大的未出芽状态。本研究进一步研究了YTM 1在细胞后期的免疫定位，发现YTM 1不仅与纺锤体微管共定位，而且与细胞质微管共定位。在去除α-因子阻断后，通过两个细胞周期在野生型酵母细胞的同步群体中检查细胞周期期间YTM 1的蛋白质水平。虽然YTM 1蛋白水平没有显著变化，但在M期YTM 1似乎略有增加。虽然YTM 1序列除了WD-40重复外没有明显的基序，但YTM 1的氨基末端显示出与RNA解旋酶和RNA聚合酶II亚基的一些相似性。YTM 1的氨基酸序列与ATP依赖性CLP蛋白酶ATP结合亚基CLPX、蛋白酶体30 kD亚基和ATP结合蛋白ABC的氨基酸序列相似性较弱，表明YTM 1在G1/S期的mRNA合成或蛋白水解以及M期纺锤体的延伸中起重要作用。

Microtubules play essential roles in a number of cellular processes such as cell motility, transport, cell shape and polarity, and mitosis. It is of importance to elucidate the mechanism which controls the organization of microtubules. We have isolated YTM1 as a multi-copy suppressor of an yeast beta-tubulin mutant in which the carboxy-terminal variable domain of the solo beta-tubulin was deleted. YTM1 encodes a novel protein of 460 amino acids with beta-transducin (WD-40) repeats. Indirect immunofluorescence method showed that YTM1 is concentrated in nuclei and also colocalizes with spindle microtubules. Co-sedimentation experiment with polymerized porcine brain tubulin showed that YTM1 can bind to microtubules. YTM1 is essential for cell growth and defect of YTM1 function causes cell cycle arrest at large unbudded state with the DNA content of G1.In this study, we examined immunolocalization more carefully and found that in late anaphase cells, YTM1 colocalized not only with spindle microtubules but also with cytoplasmic microtubules. Protein levels of YTM1 during the cell cycle were examined in a synchronized population of wild-type yeast cells through two cell cycles after removing an a-factor block. Although YTM1 protein levels did not change drastically, YTM1 appeared to be slightly increased in M phase.Although YTM1 sequence has no apparent motif except the WD-40 repeats, the amino terminus of YTM1 shows some similarity to RNA helicases and an RNA polymerase II subunit. Furthermore, the amino terminal residues shows weak similarity to the amino terminal sequence of the ATP-dependent CLP protease ATP-binding subunit CLPX,to a proteasome 30 kD subunit, and to ATP-binding protein, ABC.These sequence similarities may suggest that YTM1 plays some essential roles in mRNA production or proteolysis at the G1/S boundary as well as in spindle elongation in M phase.