Although SUMO5 facilities the growth of PML-NBs, there was heterogeneity among NBs co-expressing SUMO2/3 and SUMO5 (Fig. 6A). To differentiate the roles of SUMO5 and SUMO2/3, we used SUMO conjugation analysis and found that SUMO5 enhanced the conjugation of SUMO3 to PML (Fig. 6B), but SUMO3 conjugation promoted deconjugation of SUMO5 from PML (Fig. 6C). The polymeric SUMO chains on high-molecular-weight species of PML contained combinations of SUMO5 and SUMO3 (Fig. S6A, lane 13), reflecting a snapshot of PML-NBs of various sizes from an asynchronous cell population. It was shown that SUMO2/3 conjugation results in recruitment of the ubiquitin E3 ligase RNF4, polyubiquitination of PML and proteasome-dependent degradation of PML15,22. Therefore, we wanted to know if SUMO5 conjugation, which increased SUMO2/3 conjugation, would result in the degradation of PML-NBs. We found that RNF4 indeed mediated the disruption of SUMO5-induced PML-NBs and the dominant negative mutant RNF4(CS) blocked the RNF4-mediated degradation of PML-NBs (Fig. 6D). Furthermore, SUMO5-induced PML-NBs recruited ubiquitin (Fig. 6E) and could be preserved by the proteasome inhibitor MG132 (Fig. S6B). Moreover, RNF4 preferentially interacted with SUMO2 but not SUMO5 (Fig. 6F). These results demonstrate that SUMO5 conjugation of PML increases SUMO2/3 conjugation, which leads to the recruitment of RNF4 and ubiquitin-dependent disintegration of PML-NBs.
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