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

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 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.

SUBMITTER: Wong KH

PROVIDER: S-EPMC4220683 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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

Wong Koon Ho KH Todd Richard B RB Oakley Berl R BR Oakley C Elizabeth CE Hynes Michael J MJ Davis Meryl A MA

Fungal genetics and biology : FG & B 20080111 5

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 im ...[more]

PMID: 18262811

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Project description:We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This 'autophagosome cycle' gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS(RAB7))-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS(RAB7) or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA(RAB5)/RabB(RAB5) (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE 2-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO(RAB1). TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO(RAB1) localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO(RAB1) are dissociable by mutation: rabO(A136D) hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.

Dataset Information

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

Publications

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

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