Abstract #87C

Kar9p is restricted to one spindle pole body by two distinct phosphorylation mechanisms. Rita Miller, Jeff Moore. Dept Biol, Univ Rochester, Rochester, NY.
   During mitosis in the budding yeast Saccharomyces cerevisiae, distinct molecular properties between the two spindle poles enable them to interact differently with the polarity of the dividing cell. This enables one end of the spindle to be retained in the mother cell and the other to be transferred into the bud. The adenomatous polyposis coli (APC)-like protein Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins (MAPs) and is regulated by the yeast Cdk1, Cdc28p. Here, we show that Kar9p asymmetry is governed by two phosphorylation mechanisms, each involving a distinct cyclin and target site on Kar9p. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. We show that a phospho-mimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p targets serine 197 of Kar9p for phosphorylation. Two glutamic acid residues at positions 196 and 197 partially suppress the Kar9p asymmetry seen in the CLB4 delete. Two hybrid data suggests that modification at serine 197 attenuates the interaction between Kar9p and the XMAP215 homologue Stu2p. We propose a model in which phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear Kar9p from the mother-bound pole or transfer it onto cMTs emanating from the daughter-bound pole.

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