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

Abstract

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 singleamino-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.

Mitotic Spindle Form and Function

“…Kar9's ability to oligomerize (R. K. Miller, S.-C. Cheng, and M. D. Rose, unpublished results) may contribute to its ability to maintain persistent attachment to a dynamic substrate. Kar9 is a substrate of cyclin-dependent kinase (Liakopoulos et al 2003;Maekawa et al 2003), sumoylation (Leisner et al 2008;Meednu et al 2008), and ubiquitylation (Kammerer et al 2010). These modifications contribute to various extents to the asymmetric positioning of Kar9 on bud-directed cytoplasmic microtubules.…”

Spindle Pole Bodies Exploit the Mitotic Exit Network in Metaphase to Drive Their Age-Dependent Segregation

“…Therefore, it is the asymmetry of Kar9 distribution that promotes the alignment of the spindle with the mother-bud axis and orients the old SPB toward the bud (Liakopoulos et al, 2003; Pereira et al, 2001). Although recent studies have shed light on the regulation of Kar9 asymmetry (Liakopoulos et al, 2003; Moore et al, 2006; Moore and Miller, 2007; Leisner et al, 2008; Meednu et al, 2008; Kammerer et al, 2010; Cepeda García et al, 2010), it remains unclear how SPB identity is specified and how Kar9 asymmetry is directed toward the old SPB.…”

Spindle Pole Body History Intrinsically Links Pole Identity with Asymmetric Fate in Budding Yeast

“…Multiple posttranslational modifications have been implicated, although the involvement of CDK in particular has been the topic of contrasting reports [30][31][32][33]. Furthermore, the respective impact of those modifications on Kar9-Bim1 complex formation, its dynamics, or Kar9 polarized localization remains unclear [34][35][36]. Recently, phosphorylation by a mitotic exit network (MEN) kinase somehow linked to the MEN-specific function of Nud1 [37] was implicated in Kar9 retention by the old SPB.…”