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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Substrates Related to Chromatin and to RNA-Dependent Processes Are Modified by Arabidopsis SUMO Isoforms That Differ in a Conserved Residue with Influence on Desumoylation^1,[W],[OA]

Department of Plant Developmental Biology (R.B., R.H., K.P., G.C., A.B.) and Mass Spectrometry Group (J.S., T.C.), Max Planck Institute for Plant Breeding Research, D–50829 Cologne, Germany; Institute of Biochemistry, University of Cologne, D–50931 Cologne, Germany (S.M.); and Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, A–1030 Vienna, Austria (A.B.)

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.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andreas Bachmair (andreas.bachmair{at}univie.ac.at).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.135053

^* Corresponding author; e-mail andreas.bachmair{at}univie.ac.at.

Received December 29, 2008; accepted January 10, 2009; published January 16, 2009.

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