Lethal level overexpression of gamma-tubulin in fission yeast causes mitotic arrest.

Abstract

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.