Abstract
Proteins can be post-translationally modified by the attachment of members of the ubiquitin-like protein family which requires the action of three enzymatic steps. The protein becomes activated by an E1 enzyme, transferred to an E2 enzyme and bound to the target with the help of E3 ligases. The modification of substrates with ubiquitin-like proteins can be reversed by isopeptidases. Nedd8 and Sumo are members of this protein family. Both proteins are conserved in the model organism Aspergillus nidulans. Whereas deletion of the nedd8 homolog neddH leads to cell death, the fungus can survive SumO deficiency but displays defects in sexual and asexual development. In this work, the two-component NeddH E3 ligase DcnA/RbxA was investigated. DcnA displays in vivo interactions with the neddylation machinery. Deletion of the gene leads to a moderate reduction of cullin neddylation levels but does not have consequences on fungal development. The RING finger protein RbxA has ligase activity towards NeddH and ubiquitin. A deletion of the corresponding gene leads to cell death. In a previous study with a strain deficient in the NeddH isopeptidase CSN, developmental relevant substrate adaptors of the SCF ubiquitin E3 ligase complexes (Fbox proteins) were identified. In this work it was found that the biochemical enrichment of Fbox15 was not due to a general stabilization of the protein but likely to an arrest of a subportion of an Fbox15 containing SCF complex. In addition, the process of sumoylation was investigated in A. nidulans. Only a small subpopulation of proteins is sumoylated under normal growth conditions. To enrich SumO modified proteins, the genes for the two SumO isopeptidases UlpA and UlpB were deleted. Biochemical experiments in wild type and an UlpA deficient strain lead to the identification of a complex SumO network. This includes besides the sumoylating enzymes (E1, E2 and E3), histone modifying enzymes and complexes, transcriptional regulators, proteins involved in RNA maturation or stress response, as well as cross-talk with the processes of ubiquitination and neddylation. An interface of sumoylation and histone modification is the COMPASS complex which is involved in histone methylation. To better understand the role of the complex in the regulation of fungal development, the core subunit SetA was deleted. The resulting strain displayed defects in early sexual development, colony growth and secondary metabolism. Additionally, SetA is important for proper positioning of the asexual spore producing units.
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