Investigation Of The Mechanism For Assembly/Disassembly Of SCF Ubiquitin Ligases In Neurospora Crassa

The ubiquitin-proteasome system(UPS)is a major pathway controlling protein degradation by targeting substrate proteins for rapid ubiquitination in an E1-E2-E3 enzymatic cascade,of which the E3 ubiquitin ligases interact with substrates to catalyze ubiquitin transfer from E2 to substrates and confer specificity to the ubiquitination pathway.As the largest family of E3 ligases,the cullin-RING E3 ligases(CRLs)are multi-subunit complexes composed of a RING(Really Interesting New Gene)protein Rbxland a substrate recognition module nucleated around a cullin scaffold protein,which can be modified by ubiquitin-like protein Nedd8(Neural precursor cell Expressed Developmentally Down-regulated protein 8)on a conserved lysine residue at C-terminus to activate the CRLs.Previous data suggested a model that the CRLs assembly and activity are controlled by cycles of cullin neddylation and de-neddylation,based on the de-neddylation activity of the COP9 signalosome(CSN)and global sequestration of cullins by Cand-1(Cullin-Associated and Neddylation-Dissociated 1)binding.This prediction can rationalize the cullin neddylation cycle paradox but is hampered in recent studies,and the mechanism for this regulation and the key players remain to be identified.Here we found that,the Cullinl neddylation site mutation(cul1K722R)resulted in similar conidiation rhythm to CSN subunits knockout strains(csn-1KO,can-2KO,csn-4KO csn-5KO,csn-6K0 and csn-7KO),suggesting the importance of maintaining a proper CullNedd8/Cull ratio(un-neddylated Cull makes up a large portion of the total Cull)to ensure the normal activity of SCF.Reducing the high Cul1Nedd8/Cull ratios in csn mutants through ectopic expression of the non-neddylatable Cul1K722R proteins significantly rescues their defective phenotypes,including irregular conidiation,impaired light-regulation processes and thermo-sensitive growth.In vivo protein degradation assays reveal that,the large portion of un-neddylated Cull contributes to F-box proteins stabilization and substrates degradation.We also found that the un-neddylated Cull proteins tend to associate with the substrate adaptor modules(composed of Skp-1 and F-box proteins)but not Cand-1.And disruption of the Cull-Skp-1 binding failed to rescue the csn phenotypes as well as F-box proteins stability,demontrating that Cull-Skp-1 association is required for F-box proteins stabilization.Together,we propose that the un-neddylated Cull is another central player in the neddylation/de-neddylation cycles through the formation of Cul1-Skp-1-F-box complexes to maintain the adaptor module pool and promote rapid SCF assembly and activation,which is a new function of the un-neddylated Cull protein in cells and indicates a new mechanism for dynanic SCF organization.