Academic journal article Genetics

The Small Ubiquitin-Like Modifier (SUMO) and SUMO-Conjugating System of Chlamydomonas Reinhardtii

Academic journal article Genetics

The Small Ubiquitin-Like Modifier (SUMO) and SUMO-Conjugating System of Chlamydomonas Reinhardtii

Article excerpt

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.

POST-TRANSLATIONAL modification can regulate protein function and cellular processes in a rapid and reversible manner. In addition to protein modifi- cation by small molecules such as phosphate and carbohydrates, peptides and small proteins also serve as modifiers. The three most studied small polypeptides that covalently modify other cellular proteins are ubiquitin, small ubiquitin-like modifier (SUMO), and neural precursor cell-expressed developmentally downregulated (Nedd)8 ( Johnson 2004; Kerscher et al. 2006; Geiss-Friedlander and Melchior 2007; Palancade and Doye 2008). Ubiquitin amino acid sequence is highly conserved and the conjugation of ubiquitin to target proteins usually, but not always, results in their degradation by the 26S proteasome (Pickart 2000, 2001, 2004). Nedd8 shares high similarity with ubiquitin (60% identity and 80% similarity), and the primary substrates for Nedd8 in yeast and mammalian cells are Cullin proteins that play an important role in ubiquitin-mediated proteolysis (Kamitani et al. 1997; Yeh et al. 2000; Pan et al. 2004).

The three-dimensional (3-D) structure of human and yeast SUMO closely resembles that of ubiquitin (Melchior 2000; Hay 2001; Weissman 2001; Seeler and Dejean 2003; Johnson 2004). A prominent structural feature of SUMO is a long and highly flexible N terminus, which protrudes from the globular core of the protein. Despite the similarities in overall conformation, SUMO functions quite differently from ubiquitin. That is, SUMOylation often enables target proteins to participate in new and diverse cellular processes, including nuclear transportation, transcriptional regulation, maintenance of genome integrity, and signal transduction (Seeler and Dejean 2003; Colby et al. 2006).

In yeast and invertebrates, a single SUMO gene has been identified and has been shown to be essential for viability in Caenorhabditis elegans and Saccharomyces cerevisiae, while in Schizosacchromyces pombe,mutants lacking the single SUMO gene remain viable, but suffer severe defects in genome maintenance (Tanaka et al. 1999; Li and Hochstrasser 2003; Broday et al. 2004). Organisms have different numbers of SUMO isoforms and some SUMO isoforms appear to fulfill specialized functions. …

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