Sumoylation in Aspergillus nidulans: sumO inactivation, overexpression and live-cell imaging.
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 sumODelta 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.
Wong
Koon Ho
KH
Department of Genetics, The University of Melbourne, Grattan Street, Parkville, Vic. 3010, Australia.
Todd
Richard B
RB
Oakley
Berl R
BR
Oakley
C Elizabeth
CE
Hynes
Michael J
MJ
Davis
Meryl A
MA
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001233:Aspergillus nidulans / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018628:Gene Dosage; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D013172:Spores, Fungal / Q000254:growth & development
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D049452:Green Fluorescent Proteins