|Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
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.