In this study, we show that the Lis1 homologue Pac1p and the CLIP-170 homologue Bik1p interact with SUMO and several enzymes of the SUMO modification pathway. Pac1p also interacts with ubiquitin. We also show that Bik1p and Pac1p interact with the STUbL enzyme Nis1p–Ris1p and that the posttranslational modifications of Pac1p are controlled by this STUbL enzyme. In summary, we showed that two new classes of conserved microtubule-associated proteins interact with and are likely to be regulated by SUMO. With this work, four different spindle-positioning proteins have now been shown to interact with SUMO.
We use several different approaches to show that Pac1p interacts with SUMO. First, the two-hybrid analysis shows that Pac1p interacts with the GG but not the GA form of SUMO. This implies, but does not prove, that the interaction is due to a conjugation event. Second, inhibition of the SUMO-specific protease Ulp1p results in higher–molecular weight forms of Pac1p. This suggests that at least some of the higher–molecular weight forms are caused by SUMO moieties conjugated onto Pac1p. Indeed, when an additional SUMO is provided to the cell on a plasmid, a similar but significantly stronger banding pattern is seen. The higher–molecular weight forms of Pac1p are likely due to poly-SUMO chain formation, because the shifts are greatly diminished by the presence of a nonchainable form of SUMO. Pac1p also interacts with the STUbL enzyme Nis1p–Ris1p, an enzyme that recognizes sumoylated proteins. A pull-down assay with Pac1p suggests that the higher–molecular weight forms of Pac1p contain covalently attached SUMO. Taken together, these experiments strongly support our assertion that Pac1p is conjugated with SUMO.
Our conclusion that the interaction of Pac1p with SUMO may also involve cross-talk with ubiquitin is derived from four lines of investigation. First, we see a weak two-hybrid interaction of PAC1
with ubiquitin, encoded by UBI4
. Pac1p is also likely to be modified by ubiquitin, because higher–molecular weight forms of Pac1p can be generated by the inclusion of a plasmid encoding ubiquitin. Third, ubiquitin copurifies with Pac1p, with some shifted bands cross-reacting with both anti-ubiquitin and anti-SUMO. Fourth, Pac1p interacts with the ubiquitin ligase complex, Ris1p–Nis1p, and deletion of RIS1
results in higher–molecular weight forms of Pac1p. This is consistent with our previous finding that Kar9p and Bim1p interact with Wss1p and the same STUbL (Meednu et al., 2008
). The finding that multiple spindle-positioning proteins interact with SUMO suggests the possibility that the Nis1p–Ris1p STUbL enzyme may regulate spindle positioning.
This is the first report that a member of the Lis1 family or a member of the CAP-Gly domain family is modified by SUMO. This has significant implications for the regulation of these two classes of microtubule-associated proteins, which are widely conserved across evolution. Prior to this work, only four other microtubule-associated proteins have been shown to be modified by SUMO. These are Tau (Dorval and Fraser, 2006
; Takahashi et al., 2008
), Ndc80p (Montpetit et al., 2006
), CENP-E (Zhang et al., 2008
), and Kar9p (Leisner et al., 2008
; Meednu et al., 2008
). We also showed that the EB1 homologue Bim1p interacts with SUMO by two-hybrid analysis, but details of this interaction and whether this interaction represents an actual conjugation by SUMO remain to be elucidated (Meednu et al., 2008
). Our findings are also notable in that Pac1p was not found in any of the previous genome-wide screens for sumoylated proteins (Zhou et al., 2004
), indicating that yeast SUMO-ome may not yet be complete.
Previous reports suggest that the Kar9 pathway for spindle positioning is regulated by sumoylation (Leisner et al., 2008
; Meednu et al., 2008
). Leisner et al. (2008)
showed that the spindle-positioning defect seen in the nonsumoylatable kar9
-4K-R mutant is not as severe as the smt3-331
defect. This suggests that there are additional proteins required for spindle positioning that are also regulated by sumoylation. The findings reported here suggest that Pac1p and/or Bik1p may be the additional protein(s). Thus it is likely that both spindle-positioning pathways in yeast may be regulated by sumoylation. Future studies should elucidate whether this signal transduction system regulates each pathway separately or whether they are coordinated as a unit.
Our two-hybrid bridging data suggest that Bik1p and Pac1p are both required for their mutual interaction with SUMO and Ubc9p. However, this relationship was not observed in the in vitro shift assay using purified Bik1p. This suggests that Pac1p is not required for the in vitro shift of Bik1p. The apparent discrepancy between the two assays may be reconciled if Pac1p enhances the sumoylation of Bik1p. Consistent with this idea is our observation that Bik1p from partially fractionated extracts shifted better in vitro than purified Bik1p (unpublished data). Future work to test this hypothesis is warranted.
Furthermore, the bridging data in which Bik1p is required for the Wss1p's interaction with Pac1p (but not vice versa) suggest that Bik1p may recruit Wss1p to the Bik1p–Pac1p complex (). This is consistent with our data that Pac1p is hypermodified in a strain deleted for WSS1.
Model of Bik1p and Pac1p interaction with a STUbL and Wss1p.
We propose a model in which Pac1p sumoylation and its subsequent interaction with a STUbL promotes its degradation via the proteasome (). In this model, Wss1p aids in debranching of polysumoylated and/or polyubiquitinated Pac1p at the proteasome. Because She1p regulates the association of dynactin with dynein (Woodruff et al., 2009
), our finding that She1p inhibits the modification of Pac1p suggests the possibility that sumoylation and/or ubiquitination may regulate the interaction of dynein with its accessory proteins. In our model, She1 protein blocks the ubiquitination of Pac1p. It might also promote the proteasome-mediated degradation of modified Pac1p. We speculate that the sumoylation of Pac1p may play a role in regulating the off-loading of dynein to the cell cortex.
Several questions remain. SUMO is induced by various types of cellular stress. Future work is needed to show whether the sumoylation of these key classes of microtubule-associated proteins represents a mechanism by which microtubule-dependent processes are inactivated under conditions that are inhospitable for cell division. Furthermore, both Pac1p and Lis1 serve as adaptors of dynein. Lis1 regulates some but not all of dynein's functions. It remains to be determined whether Lis1, either in neurons or epithelial cells, is also sumoylated. Our findings have implications for how this class of dynein adaptor may regulate dynein function. Because dynein is critical to a number of fundamental processes important for life, it will be important for future studies to elucidate the widespread utility of this modification in other systems.