Abstract
The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins. However, two well-expressed isoforms differ from fungal and animal consensus in a conserved glutamine (Gln) residue situated four residues from the carboxyl terminus. We tested deviations in this position in the background of SUMO1, the isoform with the highest expression level, and found that changes do not prevent conjugation to substrate proteins in vivo. Replacement of this conserved Gln by alanine resulted in a protein that was less readily removed from a substrate by SUMO protease EARLY IN SHORT DAYS4 in an in vitro reaction and apparently led to higher levels of SUMO conjugates when expressed in vivo. We used the SUMO1 variant with the Gln-to-alanine substitution, as well as SUMO3 and SUMO5 (which carry methionine and leucine, respectively, at this position), to enrich in vivo substrates. Identification of the most abundant proteins contained in these fractions indicated that they are involved in DNA-related, or in RNA-dependent, processes, such as regulation of chromatin structure, splicing, or translation. The majority of the identified bona fide substrates contain predicted sumoylation sites. A subset of the proteins was expressed in Escherichia coli and could be sumoylated in vitro.
Highlights
The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins
SUMO is covalently linked to substrate proteins by an enzyme cascade of SUMO-activating enzyme (SAE) and SUMO-conjugating enzyme (SCE) and was shown in several cases to depend on SUMO ligases for substrate selection
Microarray data suggest low expression of SUMO4 but are not informative regarding SUMO6, -7, or -8, so it is unclear under which conditions these open reading frames are expressed
Summary
The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins. We tested deviations in this position in the background of SUMO1, the isoform with the highest expression level, and found that changes do not prevent conjugation to substrate proteins in vivo. Replacement of this conserved Gln by alanine resulted in a protein that was less readily removed from a substrate by SUMO protease EARLY IN SHORT DAYS4 in an in vitro reaction and apparently led to higher levels of SUMO conjugates when expressed in vivo. The findings that pathogens manipulate SUMO conjugation (Hotson et al, 2003; Roden et al, 2004) and that sumoylation influences innate immunity (Lee et al, 2007) imply an important role in plant-pathogen relations
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