Cloning

All eight wild type CSN subunits were cloned into a single pFBDM baculovirus transfer MultiBac vector (Berger et al., 2004). His₆-Csn5 was inserted into the first multiple cloning site (MCS1) of a pFBDM vector using NheI/XmaI and Csn1 was put into MCS2 of the same vector with BssHII/NotI. Similarly, Csn2 was inserted into a second pFBDM vector using BssHII/NotI and StrepII^2x-Csn3, containing an N-terminal PreScission-cleavable StrepII^2x-tag, using NheI/XmaI. From this plasmid the Csn2/StrepII^2x-Csn3 gene cassette was excised out with AvrII/PmeI and inserted into pFBDM^{Csn1/His6Csn5}, whose multiplication module had been linearized with BstZ17I and SpeI, yielding pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3}. A pFBDM^Csn4/Csn7b vector was generated using BssHII/NotI to insert Csn4 and NheI/XmaI for Csn7b, and the resultant gene cassette was inserted into linearized pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3}, resulting in pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3/Csn4/Csn7b}. Finally, a pFBDM^Csn6/Csn8 vector was generated using BssHII/NotI for Csn6 and NheI/XmaI for Csn8 insertion. Once again the resultant gene cassette was inserted into linearized pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3/Csn4/Csn7b}, yielding the full wild type CSN vector pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3/Csn4/Csn7b/Csn6/Csn8}. A similar cloning strategy was applied for the generation of CSN^5E76A, CSN^{5E76A, H138A}, CSN^{5E212R, D213R} and CSN^4∆N1-297, except that site-directed mutageneses were performed on pFBDM^{Csn1/His6Csn5} and pFBDM^Csn4/Csn7b respectively. CSN^5E104A and CSN^5T103I were generated with the same general approach, except that that site-directed mutagenesis and sequence validation were performed on a pCRIITOPO plasmid (Invitrogen) containing StrepII^2x-Csn5. Those mutants were then ligated into a MCS1 linearized pFBDM^Csn1 plasmid. For the production of CSN^6∆Ins2 we used co-expression from two separate viruses. To this end we applied site-directed mutagenesis on the pFBDM^Csn6/Csn8 vector to delete amino acids 174–179 in Csn6, generating pFBDM^{Csn6∆Ins2/Csn8}. The gene cassette of the latter was excised out using AvrII/PmeI and inserted into BstZ17I/SpeI linearized pFBDM^Csn4/Csn7b, yielding pFBDM^{Csn4/Csn7b/Csn6∆Ins2/Csn8}. The resultant bacmid was used together with a bacmid generated from pFBDM^{Csn1/His6-Csn5/Csn2/StrepII2x-Csn3} in order to generate two baculoviruses, which were used for co-infection to generate CSN^6∆Ins2. An analogous strategy was applied to generate CSN^{4∆N/6∆Ins2}, CSN^{5H138A/6∆Ins2} and CSN^5H138A/4∆N.

The TEV site in Rbx1 as well as mutations in the WHB domain of Cul1 were obtained by site-directed mutagenesis on the pFBDM-Cul1/Rbx1 vector described in (Enchev et al., 2010), which further contained a C-terminal sortase tag described in the next section. Cloning of Cul3/Rbx1 used in the crosslinking/mass spectrometry experiments, Nedd8-pro-peptide-StrepII^2x and StrepII^2x-Den1 are described in Orthwein et al., (2015). Recombinant bacmid and virus generation as well as protein expression proceeded as described in (Enchev et al., 2012). All genes were validated by sequencing as wild type or mutant.