Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis.
We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin. In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-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 at least 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with 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 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells. 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 beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras). The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.
Dougherty
C A
CA
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106, USA.
Sage
C R
CR
Davis
A
A
Farrell
K W
KW
D020816:Amino Acid Motifs / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005838:Genotype; D006131:Growth Inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008870:Microtubules / Q000201:enzymology*; D008938:Mitosis / Q000235:genetics*; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D013816:Thermodynamics; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
D006131:Growth Inhibitors; D014404:Tubulin; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases