Loss of Hsp70-Hsp40 Chaperone Activity Causes Abnormal Nuclear Distribution and Aberrant Microtubule Formation in M-phase of Saccharomyces cerevisiae

Abstract

The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes. To elucidate the function of the yeast cytosolic hsp70 Ssa1p <i>in vivo</i>, we characterized a<i>Saccharomyces cerevisiae ssa1</i> temperature-sensitive mutant (<i>ssa1-134</i>). After shifting to the restrictive temperature (37 °C), <i>ssa1-134</i> mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2n DNA content. We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed. Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic <i>DnaJ</i>homologue in yeast) was much weaker compared with wild-type Ssa1p. These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase. In support of this idea, a <i>ydj1</i> null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than <i>ssa1-134</i>. Furthermore, both <i>ssa1-134</i> and <i>ydj1</i> null mutant cells exhibited greater sensitivity to anti-microtubule drugs. Finally, the observation that <i>SSA1</i>and <i>YDJ1</i> interact genetically with a γ-tubulin,<i>TUB4</i>, supports the idea that they play a role in the regulation of microtubule formation.