SUMO-conjugating and SUMO-deconjugating enzymes from Arabidopsis.
Posttranslational protein modification by the small ubiquitin-like modifier (SUMO) is a highly dynamic and reversible process. To analyze the substrate specificity of SUMO-conjugating and -deconjugating enzymes from Arabidopsis (Arabidopsis thaliana), we reconstituted its SUMOylation cascade in vitro and tested the capacity of this system to conjugate the Arabidopsis SUMO isoforms AtSUMO1, 2, and 3 to the model substrate ScPCNA from yeast (Saccharomyces cerevisiae). This protein contains two in vivo SUMOylated lysine residues, namely K127 and K164. Under in vitro conditions, the Arabidopsis SUMOylation system specifically conjugates all tested SUMO isoforms to lysine-127, but not to lysine-164, of ScPCNA. The SUMO isoforms AtSUMO1 and AtSUMO2, but not AtSUMO3, were found to form polymeric chains on ScPCNA due to a self-SUMOylation process. In a complementary approach, we analyzed both the SUMO isopeptidase activity and the pre-SUMO-processing capacity of the putative Arabidopsis SUMO proteases At1g60220, At1g10570, and At5g60190 using the known SUMO isopeptidases ScULP1, XopD, and ESD4 (At4g15880) as reference enzymes. Interestingly, At5g60190 exhibits no SUMO protease activity but processes the pre-form of Arabidopsis Rub1. The other five enzymes represent SUMO isopeptidases that show different substrate preferences. All these enzymes cleave AtSUMO1 and AtSUMO2 conjugates of ScPCNA, whereas only the putative bacterial virulence factor XopD is able to release AtSUMO3. In addition, all five enzymes cleave pre-AtSUMO1 and pre-AtSUMO2 peptides, but none of the proteins efficiently produce mature AtSUMO3 or AtSUMO5 molecules from their precursors.
Colby
Thomas
T
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
Matthäi
Anett
A
Boeckelmann
Astrid
A
Stuible
Hans-Peter
HP
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics; D018745:Genome, Plant; D006801:Humans; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D016959:Xanthomonas campestris / Q000201:enzymology
D029681:Arabidopsis Proteins; D018809:Proliferating Cell Nuclear Antigen; C477512:SUM1 protein, Arabidopsis; C477513:SUM2 protein, Arabidopsis; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease