Mutation in the -Tubulin Signature Motif Suppresses Microtubule GTPase Activity and Dynamics, and Slows Mitosis
详细信息 查看全文
- 作者：Cynthia A. Dougherty ; Carleton R. Sage ; Ashley Davis ; and Kevin W. Farrell
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：December 25, 2001
- 年：2001
- 卷：40
- 期：51
- 页码：15725 - 15732
- 全文大小：90K
- 年卷期：v.40,no.51(December 25, 2001)
- ISSN：1520-4995
文摘
We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signaturemotif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae 
-tubulin. In an electrondiffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotidebound in the -tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by atleast 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shorteningdynamics of MTs assembled from 
:Thr143Gly-mutated dimers were also strongly suppressed, comparedto control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistentwith the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 ± 0.2 
m, approximately half of the size of MT arrays in large-buddedwild-type cells (average length = 7.1 ± 0.4 m), suggesting that there is a delay in mitosis in the mutantcells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures(30% versus 12% for wild-type cells), again suggesting such a delay. The results show that :Thr143 ofthe tubulin signature motif plays an important role in GTP binding and hydrolysis by the -tubulin E siteand support the idea that tubulins belong to a family of proteins within the GTPase superfamily that arestructurally distinct from the classic GTPases, such as EF-Tu and p21ras. The data also suggest that MTdynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly couldbe coordinated with other cell-cycle events by regulating -tubulin GTPase activity.
-tubulin. In an electrondiffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotidebound in the -tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by atleast 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shorteningdynamics of MTs assembled from :Thr143Gly-mutated dimers were also strongly suppressed, comparedto control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistentwith the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 ± 0.2 m, approximately half of the size of MT arrays in large-buddedwild-type cells (average length = 7.1 ± 0.4 m), suggesting that there is a delay in mitosis in the mutantcells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures(30% versus 12% for wild-type cells), again suggesting such a delay. The results show that :Thr143 ofthe tubulin signature motif plays an important role in GTP binding and hydrolysis by the -tubulin E siteand support the idea that tubulins belong to a family of proteins within the GTPase superfamily that arestructurally distinct from the classic GTPases, such as EF-Tu and p21ras. The data also suggest that MTdynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly couldbe coordinated with other cell-cycle events by regulating -tubulin GTPase activity.