Originally published as Genetics Published Articles Ahead of Print on October 1, 2008.

Genetics, Vol. 180, 2033-2055, December 2008, Copyright © 2008
doi:10.1534/genetics.108.095042

The Spindle Positioning Protein Kar9p Interacts With the Sumoylation Machinery in Saccharomyces cerevisiae

Nida Meednu^*, Harold Hoops^*,, Sonia D'Silva^*, Leah Pogorzala^*, Schuyler Wood, David Farkas^*, Mark Sorrentino^*, Elaine Sia^*, Pam Meluh and Rita K. Miller^*,,1

^* Department of Biology, University of Rochester, Rochester, New York 14627, Department of Biology, State University of New York, Geneseo, New York 14454, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078

¹ Corresponding author: 258A Noble Research Center, Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078.
E-mail: rita.miller{at}okstate.edu

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.

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