The TOG protein Stu2 is regulated by acetylation.

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB.

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

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.

Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins.

Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule-associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin-regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non-covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO-targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed.