Vol. 12, Issue 9, 2672-2687, September 2001

Single Site -Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase in Saccharomyces cerevisiae: Possible Role for Palmitoylation of -Tubulin

Joan M. Caron,^* Leticia R. Vega, James Fleming, Robert Bishop,^§ and Frank Solomon

 ^*Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030;  Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and  ^§Microbial Expression, Chiron Corporation, Emeryville, California 94608

We generated a strain of Saccharomyces cerevisiae in which the sole source of -tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S -tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15°C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in -tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated -tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated -tubulin in C377S tub1 cells. Our results suggest that cys-377 of -tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.