Abstract #11

Kar9p and Bim1p interact with Smt3p and the machinery for sumoylation. Nida Meednu¹, Harold Hoops², Leah Pogorzala¹, Elaine Sia¹, Rita Miller¹. 1) Dept Biol, Univ Rochester, Rochester, NY; 2) Dept Biol, State University of New York, Geneseo, NY.
   Kar9p plays an important role in positioning the mitotic spindle by serving as a linker between the actin and microtubule cytoskeletons. The microtubule-to-Kar9p linkage occurs through Bim1p, a MT-binding protein. Kar9p localizes to the SPB and attached microtubules that are destined for the bud. However, little is known about the molecular mechanisms that regulate Kar9p function. SUMO/Smt3p is a conserved small ubiquitin-related modifier that is covalently attached to other proteins as a post-translational modification. SUMO is analogous to ubiquitin in several ways, but functionally different. Unlike ubiquitin, sumoylation does not target proteins for degradation. Although targets of sumoylation are associated with a diverse set of biological processes, it was not previously known whether sumoylation regulates the microtubule-dependent process of spindle positioning. Here, we show that Kar9p and Bim1p are likely to be modified by SUMO/Smt3p. Both interact with SMT3 by two-hybrid analysis. However, neither interact with a mutant form of SMT3 in which the terminal glycine required for conjugation has been mutated to alanine. Both Kar9p and Bim1p also interact with proteins involved in the sumoylation pathway, UBC9 an E2 enzyme required for SUMO conjugation; NFI1 an E3 conferring specificity for SUMO targets; and WSS1 a weak suppressor of SUMO. The Kar9p-Ubc9p interaction was confirmed in vitro by affinity chromatography. A single point mutation in KAR9, kar9-L304P, disrupts its two-hybrid interaction with SMT3, UBC9, and WSS1 but retains its interactions with other proteins important for spindle positioning, BIM1, MYO2, BIK1, and STU2. The mitotic spindle in the kar9-L304P mutant is mis-positioned relative to the bud neck. kar9-L304P-GFP localizes to both SPBs, rather than just the one destined for the bud. Combined, these data suggest that sumoylation may be a new mechanism for the regulation of Kar9p and Bim1p function during spindle positioning.

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