Assembly pathway of the fungal COP9 signalosome
von Fruzsina Erzsébet Bakti
Datum der mündl. Prüfung:2022-02-28
Erschienen:2022-12-20
Betreuer:Prof. Dr. Gerhard Braus
Gutachter:Prof. Dr. Kai Heimel
Gutachter:Prof. Dr. Ralf Ficner
Dateien
Name:Dissertation_Assembly pathway of the fungal ...pdf
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Zusammenfassung
Englisch
The conserved eight-subunit COP9 signalosome (CSN complex) controls the exchange of E3 ubiquitin cullin RING ligase receptors, and therefore the specificity of protein degradation in eukaryotic cells and it is required for multicellular development. The CSN complex of the filamentous fungus Aspergillus nidulans assembles through a seven-subunit pre-CSN complex, which is activated by the final integration of the catalytic CsnE deneddylase, thereby forming an eight-subunit CSN, as in humans. The goal of this work was to identify the assembly pathway of fungal pre-CSN complex in vivo by combined genetic and biochemical approaches. Loss of CsnE and the abolishment of the pre-CSN complex formation result in distinct fungal phenotypes. All fungal COP9 signalosome subunits are required for sexual development and secondary metabolism. Subunits of the pre-CSN complex additionally support wild type-like growth and asexual development. Functional GFP-CSN subunit fusions can complement the corresponding phenotypes. GFP-CSN subunit-based GFP-affinity enrichments combined with mass spectrometry allowed the identification of interacting CSN subcomplexes. The interactome analysis revealed, as one major result, that two distinct heterotrimeric CSN subcomplexes are intermediates of the assembly pathway. These heterotrimers, CsnA-CsnC-CsnH and CsnD-CsnF-CsnG, are formed independently of CsnB. CsnE cannot associate to either any of the heterotrimers or CSN subunits without CsnB, which supports that this subunit acts as link between the two trimeric subcomplexes. Subtle cellular feedback surveillance mechanisms control that equal CSN subunit amounts are available for CSN assembly. Accordingly, loss of csn subunit genes differentially changes protein levels of remaining CSN subunits. Subunits of both heterotrimeric subcomplexes are required to reach wild type-like CsnA protein amounts, which is part of the CsnA-CsnC-CsnH subcomplex. In contrast, relative protein levels of CsnD, which is part of the CsnD-CsnF-CsnG trimer, are increased, if the pre-CSN complex cannot be formed. CsnG is a prerequisite to connect subunits of the CsnD-CsnF-CsnG subcomplex and it is part of the surveillance for the cellular CsnD amounts. Furthermore, CsnA and, therefore the CsnA-CsnC-CsnH trimer, is required for appropriate CsnB amounts, because the inability to form CsnA-CsnC-CsnH reduces relative levels of the CsnB bridging protein. These results support a complex orchestration for providing sufficient levels of subunits for the correct CSN assembly pathway, which includes a mutual control of the formation of both trimers. In addition, the cellular abundance of the CsnE deneddylase and the CsnB linker are adjusted independently of each other. An additional level of control includes the cellular localization of CSN subunits. The stable octameric CSN complex is enriched in nuclei, whereas CSN subassemblies inhabit different cellular compartments. The CsnD-CsnF-CsnG trimer is predominantly nuclear localized, whereas CsnA-CsnC-CsnH is evenly distributed between nuclei and cytoplasm. Nuclear enrichment of the linker CsnB is independent of the deneddylase subunit, but depends on CsnA and, therefore, an intact CsnA-CsnC-CsnH trimer. This suggests that CsnB is transported into the nuclei together with CsnA. In summary, the fungal COP9 signalosome is assembled through two trimeric subcomplexes linked by CsnB, which enables final association with the CsnE deneddylase. Formation of the functional CSN complex requires a sophisticated interplay between mutual control of subunit protein levels, correct localization and sequential assembly.
Keywords: COP9 signalosome, CSN complex, pre-CSN complex, native CSN subcomplexes, assembly, affinity purification, subcellular localization, Aspergillus nidulans