Lethal level overexpression of gamma-tubulin in fission yeast causes mitotic arrest.
gamma-Tubulin is a member of the tubulin superfamily and plays essential roles in microtubule nucleation. While the level of other tubulins, alpha- and beta-tubulin, is strictly regulated in higher eukaryotes and overexpression of beta-tubulin is toxic in yeasts, gamma-tubulin can be overexpressed by fivefold in fission yeast without any obvious defect in growth. Extreme overexpression of gamma-tubulin in mammalian cells caused growth arrest; however, the exact level of gamma-tubulin and the critical level of gamma-tubulin necessary for growth defect were undetermined. We have constructed strains that over- or underexpress gamma-tubulin by placing the gamma-tubulin gene under the control of the inducible nmt1 promoter and its variants. Among these, the weakest promoter was able to produce enough gamma-tubulin to support normal growth when its expression was induced. A strain in which the gamma-tubulin gene was placed under the control of the strongest inducible promoter achieved 160-fold overexpression of gamma-tubulin and its growth was suppressed. Normal cytoplasmic microtubules were mostly lost in gamma-tubulin overexpressing cells and gamma-tubulin was accumulated around the periphery of nuclei. Many of the cells were arrested in mitosis. A small fraction of cells did proceed to undergo nuclear division; however, its process looked either significantly deterred or abnormal. Our results presented here suggest that excess gamma-tubulin disrupts the microtubule array and significantly deters the formation of the mitotic spindle, most likely because of random nucleation of microtubules from excess gamma-tubulin in the cytoplasm.
Horio
T
T
Department of Food Microbiology, The University of Tokushima School of Medicine, Kuramoto, Tokushima, Japan. horio@nutr.med.tokushima-u.ac.jp
Basaki
A
A
Takeoka
A
A
Yamato
M
M
D015153:Blotting, Western; D002453:Cell Cycle; D016923:Cell Death / Q000235:genetics; D018385:Centrosome / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008938:Mitosis; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics*
D018076:DNA, Complementary; D014404:Tubulin