Arabidopsis TCP Transcription Factors Interact with the SUMO Conjugating Machinery in Nuclear Foci

Abstract

In Arabidopsis more than 400 proteins have been identified as SUMO targets, both in vivo and in vitro. Among others, transcription factors (TFs) are common targets for SUMO conjugation. Here we aimed to exhaustively screen for TFs that interact with the SUMO machinery using an arrayed yeast two-hybrid library containing more than 1,100 TFs. We identified 76 interactors that foremost interact with the SUMO conjugation enzyme SCE1 and/or the SUMO E3 ligase SIZ1. These interactors belong to various TF families, which control a wide range of processes in plant development and stress signaling. Amongst these interactors, the TCP family was overrepresented with several TCPs interacting with different proteins of the SUMO conjugation cycle. For a subset of these TCPs we confirmed that the catalytic site of SCE1 is essential for this interaction. In agreement, TCP1, TCP3, TCP8, TCP14, and TCP15 were readily SUMO modified in an E. coli sumoylation assay. Strikingly, these TCP-SCE1 interactions were found to redistribute these TCPs into nuclear foci/speckles, suggesting that these TCP foci represent sites for SUMO (conjugation) activity.

The SUMO conjugation complex self-assembles into nuclear bodies independent of SIZ1 and COP1

“…S1G), we conclude that SIM(-like) interactions also play a role in the SUMO-SCE1 interaction. Additional support comes from our recent work where we demonstrated that a conserved SIM-like motif in the N-terminal tail of SCE1 is essential for SUMO1 binding (Mazur et al, 2017). Our findings here suggest the same for SUMO-SIZ1 protein complexes in Arabidopsis, as previously reported for the homologous proteins from yeast and mammals (Cheong et al, 2010; Mascle et al, 2013; Kaur et al, 2017); however, functional mutants of the SIM domain in SIZ1 still need to be tested.…”

“…Also in the BiFC assay, both the SCE1 SUM1 and SCE1 SIZ1 variants failed to interact with SIZ1, while the catalytic-dead variants (SCE1 CAT1 and SCE1 CAT2 ) still interacted with SIZ1. This suggests that SUMO loading in the catalytic site of SCE1 (SCE1 CAT1 and CAT2 ; E2∼ SUMO1) is not required for SCE1 and SIZ1 to interact, while the non-covalent interaction (SCE1 SUM1 ; E2 · SUMO1) strengthens this interaction in both the Y2H and BiFC assays (Fig 1F; Mazur et al, 2017). Apparently, the non-covalently bound SUMO acts as a glue in the SCE1-SIZ1 complex or it alters the SCE1 conformation such that it enhances the interaction between SCE1 and SIZ1.…”

“…To further discern how these NBs are formed, different Arabidopsis SCE1 variants were used that (i) cannot interact with SUMO or SIZ1, (ii) had lost their catalytic activity (SCE1 CAT1 and CAT2 ), or (iii) no longer recognized the ψKxE SUMO acceptor motif in SUMO substrates (SCE1 CAT2 ) (Mazur et al, 2017) (Fig. 1D; Supplemental Table S1).…”

“…These five residues are strictly conserved in Arabidopsis SCE1 and other plant homologues of SCE1 (Supplemental Fig. S4), and also for Arabidopsis SCE1 these residues are essential for its interaction with SUMO1 or SIZ1 in the Y2H assay (Mazur et al, 2017). The SCE1 CAT1 mutant still interacted with SUMO1; however, SCE1 CAT2 had lost both its catalytic activity and its non-covalent interaction with SUMO1 (Mazur et al, 2017).…”

“…S3F). We already showed that the SCE1 residues Pro70 and Pro106 (Supplemental Table S1, SCE1 SIZ1 ) are essential for SCE1 and SIZ1 to interact in the Y2H assay, while mutating the catalytic site (SCE1 CAT1 and SCE1 CAT2 ) did not impair their Y2H interaction (Mazur et al, 2017). Also in the BiFC assay, both the SCE1 SUM1 and SCE1 SIZ1 variants failed to interact with SIZ1, while the catalytic-dead variants (SCE1 CAT1 and SCE1 CAT2 ) still interacted with SIZ1.…”