The Small Ubiquitin-Like Modifier (SUMO) and SUMO-Conjugating System ofChlamydomonas reinhardtii

Abstract

The availability of the complete DNA sequence of the Chlamydomonas reinhardtii genome and advanced computational biology tools has allowed elucidation and study of the small ubiquitin-like modifier (SUMO) system in this unicellular photosynthetic alga and model eukaryotic cell system. SUMO is a member of a ubiquitin-like protein superfamily that is covalently attached to target proteins as a post-translational modification to alter the localization, stability, and/or function of the target protein in response to changes in the cellular environment. Three SUMO homologs (CrSUMO96, CrSUMO97, and CrSUMO148) and three novel SUMO-related proteins (CrSUMO-like89A, CrSUMO-like89B, and CrSUMO-like90) were found by diverse gene predictions, hidden Markov models, and database search tools inferring from Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana SUMOs. Among them, CrSUMO96, which can be recognized by the A. thaliana anti-SUMO1 antibody, was studied in detail. Free CrSUMO96 was purified by immunoprecipitation and identified by mass spectrometry analysis. A SUMO-conjugating enzyme (SCE) (E2, Ubc9) in C. reinhardtii was shown to be functional in an Escherichia coli-based in vivo chimeric SUMOylation system. Antibodies to CrSUMO96 recognized free and conjugated forms of CrSUMO96 in Western blot analysis of whole-cell extracts and nuclear localized SUMOylated proteins with in situ immunofluorescence. Western blot analysis showed a marked increase in SUMO conjugated proteins when the cells were subjected to environmental stresses, such as heat shock and osmotic stress. Related analyses revealed multiple potential ubiquitin genes along with two Rub1 genes and one Ufm1 gene in the C. reinhardtii genome.

SUMOylation by a Stress-Specific Small Ubiquitin-Like Modifier E2 Conjugase Is Essential for Survival ofChlamydomonas reinhardtiiunder Stress Conditions

“…CrUBC3 Is Most Likely the Second SUMO E2 Conjugase Gene in C. reinhardtii Previous analyses of potential SUMO E2 conjugases in C. reinhardtii (Wang et al, 2008) suggested that, among the candidate genes, CrUBC9 was the most similar to verified SUMO E2 conjugase genes found in other eukaryotic organisms. In that same analysis, however, it was noted that the distinction between some of the candidate enzymes as SUMO conjugases versus ubiquitin conjugases was uncertain (Wang et al, 2008).…”

“…It was previously shown that multiple high-M r proteins are SUMOylated in C. reinhardtii in response to a heat shock of 428C (Wang et al, 2008). To determine if mut5, which lacks the CrUbc9 gene, is capable of this stress-induced SUMOylation, wild-type and mut5 cultures were shifted to 428C for 1 h, and the patterns of SUMOylation in these cultures were detected and analyzed on immunoblots using anti-SUMO antibodies (Fig.…”

“…In our initial characterization of the SUMOylation system of Chlamydomonas reinhardtii, we identified a protein named CrUBC9 as the most likely SUMO E2 conjugase in this organism and showed that it possessed SUMO E2 conjugase activity in vitro (Wang et al, 2008). Additionally, it was shown by immunoblot analysis that SUMOylation patterns markedly changed in response to various abiotic stress conditions (Wang et al, 2008).…”

“…Additionally, it was shown by immunoblot analysis that SUMOylation patterns markedly changed in response to various abiotic stress conditions (Wang et al, 2008). Because in virtually every other haploid or diploid organism studied, a single SUMO E2 conjugase had been identified, it was presumed that CrUBC9 was likely the only SUMO E2 conjugase present in the C. reinhardtii genome and that it was essential for cell viability.…”

Effect of Cu content on the activity of Cu/ZnSOD1 in the Arabidopsis SUMO E3 ligasesiz1mutant

“…The components of SUMO conjugation and deconjugation systems are observed in many plant species, including algae, dicots and monocots. [1][2][3] SUMOylation is involved in many plant physiological processes, such as controlling cell growth and development, embryogenesis and flowering time regulation. 4 It is also involved in actions of both biotic and abiotic stresses, including phosphate starvation responses, salicylic acid-dependent pathogen defense, freezing and cold tolerance, basal thermotolerance and drought response.…”