Sumoylation in Aspergillus nidulans: sumO inactivation, overexpression and live-cell imaging

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Abstract

Sumoylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. In Saccharomyces cerevisiae, but not in Schizosaccharyomes pombe, deletion of the gene encoding SUMO peptides is lethal. We have characterized the SUMO-encoding gene, sumO, in the filamentous fungus Aspergillus nidulans. The sumO gene was deleted in a diploid and sumOΔ haploids were recovered. The mutant was viable but exhibited impaired growth, reduced conidiation and self-sterility. Overexpression of epitope-tagged SumO peptides revealed multiple sumoylation targets in A. nidulans and SumO overexpression resulted in greatly increased levels of protein sumoylation without obvious phenotypic consequences. Using five-piece fusion PCR, we generated a gfp-sumO fusion gene expressed from the sumO promoter for live-cell imaging of GFP-SumO and GFP-SumO-conjugated proteins. Localization of GFP-SumO is dynamic, accumulating in punctate spots within the nucleus during interphase, lost at the onset of mitosis and re-accumulating during telophase.

Introduction

Sumoylation, the covalent addition of a small ubiquitin-like modifier (SUMO) peptide, is a conserved post-translational modification that can influence the activity, interaction or subcellular location of a diverse array of proteins (Melchior, 2000, Verger et al., 2003). The addition of SUMO peptides to target proteins is catalyzed by a series of enzymatic steps in which the SUMO peptide precursor is first cleaved to produce a mature peptide with a C-terminal di-glycine motif (Johnson et al., 1997, Kamitani et al., 1997). SUMO peptides are activated by the E1-activating enzyme and transferred to the E2-conjugating enzyme Ubc9 for covalent attachment, via the di-glycine motif, to a lysine residue on the target protein by a member of the E3-SUMO ligase family (Johnson and Blobel, 1997, Schwarz et al., 1998, Takahashi et al., 2001). The covalent attachment of SUMO peptides to proteins is reversible by a family of SUMO-specific proteases (Gong et al., 2000, Gong and Yeh, 2006, Li and Hochstrasser, 1999, Yeh et al., 2000).

In higher eukaryotes, sumoylation is involved in cell cycle progression, genome stability, DNA repair, transcriptional regulation and signal transduction (see Gill, 2004, Johnson, 2004, Verger et al., 2003 for reviews). Proteomic analyses in Saccharomyces cerevisiae has revealed that sumoylation is involved in a similarly broad range of functions including transcription, translation, DNA replication and stress responses (Denison et al., 2005, Hannich et al., 2005, Panse et al., 2004, Wohlschlegel et al., 2004, Zhou et al., 2004). SUMO peptides are a highly conserved family of proteins found in all eukaryotes. In vertebrates, the SUMO family comprises four genes while eight SUMO genes have been identified in Arabidopsis thaliana (Bohren et al., 2004, Kurepa et al., 2003, Su and Li, 2002). In contrast, the yeasts S. cerevisiae and Schizosaccharomyces pombe each contain a single gene (SMT3 and pmt3, respectively) (Giaever et al., 2002, Johnson et al., 1997, Tanaka et al., 1999). Deletion of SMT3 in S. cerevisiae is lethal whereas the pmt3Δ mutant in S. pombe is viable but shows severely reduced growth and aberrant cellular and nuclear morphologies (Giaever et al., 2002, Johnson et al., 1997, Tanaka et al., 1999).

We have identified the sumO gene encoding the SUMO peptide in Aspergillus nidulans and show that inactivation of this gene is not lethal in this filamentous fungus. The role of sumoylation has been investigated by determining the in vivo effects of gene inactivation and of sumO overexpression. The tagging of the SUMO peptide with GFP has revealed a dynamic pattern to the subcellular localization of sumolyated proteins during the cell cycle.

Section snippets

Fungal strains, media and molecular methods

Complete and ANM minimal media were as described by Cove, 1966 and YAG medium contained 5 g/L of yeast extract and 20 g/L d-glucose. Minimal medium contained 1% glucose or xylose where indicated to induce expression from the xylP promoter. Nitrogen sources were added at a concentration of 10 mM unless otherwise indicated. For testing sensitivity to compounds, 0.0025% methyl methanesulfonate (MMS) or 5 mM hydroxyurea (HU) was added to glucose minimal medium containing ammonium tartrate as the

A single SUMO peptide-encoding gene is present in A. nidulans

A single gene (AN1191.3), designated sumO, was identified in the A. nidulans genome sequence using the Blastp algorithm for sequences predicted to encode a SUMO-1-like peptide. The sumO gene was PCR amplified from genomic DNA, cloned and sequenced (Section 2). Comparison of the sumO genomic sequence and a full-length sumO EST (GenBank Accession No. AAB01675) revealed a single 98 bp intron (+223 to +320). The predicted sumO gene product of 94 amino acids shows considerable similarity to S.

Discussion

An incredibly diverse range of cellular proteins serve as substrates for the covalent attachment of SUMO peptides in vertebrates and in yeast (e.g. Hannich et al., 2005, Wohlschlegel et al., 2004). It is not clear how sumoylation affects the various cellular processes in which it is involved and the consequences of SUMO attachment vary between substrates. The covalent linkage of SUMO peptides can directly affect the activity of target proteins by altering their protein or DNA interactions,

Acknowledgments

We acknowledge the support of the Australian Research Council, the award of an International Postgraduate Research Scholarship (IPRS) and Melbourne International Research Scholarship (MIRS) to Koon Ho Wong, and the support of the National Institute of General Medical Sciences (Grant GM031837) to Berl Oakley. We thank K. Nguyen for expert technical assistance and Yi Xiong and Dr. Edyta Szewczyk, The Ohio State University, for construction of mCherry γ-tubulin. We acknowledge support from the

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