Normal microtubule function and the interaction between the pathways for tubulin folding and expression in S. cerevisiae

Abstract

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.Includes bibliographical references.Undimerized -tubulin is toxic to the yeast Saccharomyces cereivisiae. Free P-tubulin can arise if the tubulin heterodimer dissociates or if levels of 3-tubulin and cc-tubulin are unbalanced. I am using the toxicity of 3-tubulin to understand the early steps in microtubule morphogenesis. I have found that a mutation of the gene PLP1 allows cells to survive the toxicity of [3-tubulin produced from disparate levels of - and -tubulin. The suppression occurs either when c-tubulin is modestly underexpressed relative to -tubulin, or when 13-tubulin is inducibly and strongly overexpressed. A significant proportion of the undimerized -tubulin in plpl cells is less toxic and less functional than in wild type cells. As a result, piplA cells have lower levels of heterodimer. Significantly, plpl cells that also lack PaclOp, a component of the GimC/Pfd complex that helps fold tubulin polypeptides, are even less affected by free -tubulin. Our results suggest that Plplp defines a novel step in -tubulin folding. My work demonstrates an interaction between the pathways for tubulin folding and the regulation of tubulin expression. Cells that are paclOA piplA have much less folded and functional -tubulin than even plplA cells, and also upregulate -tubulin through increasing transcription. The upregulation of -tubulin RNA is dependent on the putative transcription factor Cin5p. In the absence of CIN5, paclO plpl A cells have decreased tubulin heterodimer levels, down to approximately 20% that of wild type. The heterodimer levels are also decreased from paclO plpl cells suggesting that the limiting factor in heterodimer formation in paclO plpl in5A is -tubulin. The paclOA plpl cin5 cells grow normally, but have mitotic defects such as abnormal nuclear positioning and short anaphase spindles.(cont.) The Cin5p dependent upregulation of 3-tubulin may be a mechanism to maintain tubulin heterodimer levels and so sustain normal microtubule function.by Soni Lacefield Shimoda.Ph.D