RESUMEN
DenA/DEN1 and the COP9 signalosome (CSN) represent two deneddylases which remove the ubiquitin-like Nedd8 from modified target proteins and are required for distinct fungal developmental programmes. The cellular DenA/DEN1 population is divided into a nuclear and a cytoplasmatic subpopulation which is especially enriched at septa. DenA/DEN1 stability control mechanisms are different for the two cellular subpopulations and depend on different physical interacting proteins and the C-terminal DenA/DEN1 phosphorylation pattern. Nuclear DenA/DEN1 is destabilized during fungal development by five of the eight CSN subunits which target nuclear DenA/DEN1 for degradation. DenA/DEN1 becomes stabilized as a phosphoprotein at S243/S245 during vegetative growth, which is necessary to support further asexual development. After the initial phase of development, the newly identified cytoplasmatic DenA/DEN1 interacting phosphatase DipA and an additional developmental specific C-terminal phosphorylation site at serine S253 destabilize DenA/DEN1. Outside of the nucleus, DipA is co-transported with DenA/DEN1 in the cytoplasm between septa and nuclei. Deletion of dipA resulted in increased DenA/DEN1 stability in a strain which is unresponsive to illumination. The mutant strain is dysregulated in cytokinesis and impaired in asexual development. Our results suggest a dual phosphorylation-dependent DenA/DEN1 stability control with stabilizing and destabilizing modifications and physical interaction partner proteins which function as control points in the nucleus and the cytoplasm.
Asunto(s)
Aspergillus nidulans/genética , Endopeptidasas/genética , Proteínas Fúngicas/genética , Monoéster Fosfórico Hidrolasas/genética , Procesamiento Proteico-Postraduccional , Ubiquitinas/metabolismo , Aspergillus nidulans/crecimiento & desarrollo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocinesis/genética , Citoplasma/enzimología , Endopeptidasas/metabolismo , Escherichia coli , Regulación Fúngica de la Expresión Génica , Complejos Multiproteicos , Fosforilación , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ubiquitina , Ubiquitinas/genéticaRESUMEN
Deneddylases remove the ubiquitin-like protein Nedd8 from modified proteins. An increased deneddylase activity has been associated with various human cancers. In contrast, we show here that a mutant strain of the model fungus Aspergillus nidulans deficient in two deneddylases is viable but can only grow as a filament and is highly impaired for multicellular development. The DEN1/DenA and the COP9 signalosome (CSN) deneddylases physically interact in A. nidulans as well as in human cells, and CSN targets DEN1/DenA for protein degradation. Fungal development responds to light and requires both deneddylases for an appropriate light reaction. In contrast to CSN, which is necessary for sexual development, DEN1/DenA is required for asexual development. The CSN-DEN1/DenA interaction that affects DEN1/DenA protein levels presumably balances cellular deneddylase activity. A deneddylase disequilibrium impairs multicellular development and suggests that control of deneddylase activity is important for multicellular development.
Asunto(s)
Aspergillus nidulans , Endopeptidasas , Complejos Multiproteicos , Péptido Hidrolasas , Ubiquitinas , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Complejo del Señalosoma COP9 , Endopeptidasas/genética , Endopeptidasas/metabolismo , Regulación Fúngica de la Expresión Génica , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteína NEDD8 , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.