The TOG protein Stu2/XMAP215 interacts covalently and noncovalently with SUMO

Abstract

Stu2p is the yeast member of the XMAP215/Dis1/ch-TOG family of microtubule-associated proteins that promote microtubule polymerization. However, the factors that regulate its activity are not clearly understood. Here we report that Stu2p in the budding yeast Saccharomyces cerevisiae interacts with SUMO by covalent and noncovalent mechanisms. Stu2p interacted by two-hybrid analysis with the yeast SUMO Smt3p, its E2 Ubc9p, and the E3 Nfi1p. A region of Stu2p containing the dimerization domain was both necessary and sufficient for interaction with SUMO and Ubc9p. Stu2p was found to be sumoylated both in vitro and in vivo. Stu2p copurified with SUMO in a pull-down assay and vice versa. Stu2p also bound to a nonconjugatable form of SUMO, suggesting that Stu2p can interact noncovalently with SUMO. In addition, Stu2p interacted with the STUbL enzyme Ris1p. Stu2p also copurified with ubiquitin in a pull-down assay, suggesting that it can be modified by both SUMO and ubiquitin. Tubulin, a major binding partner of Stu2p, also interacted noncovalently with SUMO. By two-hybrid analysis, the beta-tubulin Tub2p interacted with SUMO independently of the microtubule stressor, benomyl. Together, these findings raise the possibility that the microtubule polymerization activities mediated by Stu2p are regulated through sumoylation pathways.

How Does SUMO Participate in Spindle Organization?

“…This is in accordance with the observed presence of SUMO1 at spindle poles and SUMO2/3 at chromosomes/kinetochores [36]. On the spindle, the yeast MAP protein Stu2/XMAP215 was identified as SUMO conjugate [77], as well as the yeast spindle positioning factor Kar9 [67,80]. The number of SUMOylated spindle proteins will very likely increase, since several MAPs are found to be SUMOylated within the SUMOylome proteomic data (see §4 and Table 2).…”

The TOG protein Stu2 is regulated by acetylation

“…Tween, 20 mM N-Ethylmaleimide, 20 mM 2-Iodoacetamide, 1:500 Sigma protease inhibitor cocktail #8849, and 1 mM PMSF, and clarified as described in Greenlee et. al., 2018.…”

“…Yeast cells were grown to an OD600 value of 0.3 and treated with a final concentration of 200 ng/mL TSA in DMSO or DMSO alone for 2 hr at 30 °C. Following TSA treatments, cells were harvested, cryo-milled, resuspended in 1x PBS containing 0.1% Tween, 20 mM N-Ethylmaleimide, 20 mM 2-Iodoacetamide, 1:500 Sigma protease inhibitor cocktail #8849, and 1 mM PMSF, and clarified as described in Greenlee et. al., 2018.…”

“…Yeast expressing Stu2-HA (yRM10637) or a vector control (yRM10641) were grown to mid-exponential phase and disrupted using cryo-milling as described in Greenlee et al, 2018. Frozen powdered cell debris, termed "fluff," was resuspended in 1x PBS containing 0.1%…”

“…Phosphorylation of other XMAP215 family members has also been reported to control their association with the microtubule lattice; in S. pombe CDK mediated phosphorylation was found in Alp14 (Aoki et al, 2006) and Dis1 proteins (Okada et al, 2014), and Msps phosphorylation was found in D. melanogaster (Trogden & Rogers, 2015). While phosphorylation of its MT-binding domain regulates Stu2 association with the microtubule lattice (Humphrey et al, 2018) (Okada et al, 2014) (Trogden & Rogers, 2015), the role that other posttranslational modifications such as sumoylation play in governing Stu2 localization and functions remain elusive (Greenlee et al, 2018).…”