Tandem affinity purification and mass spectrometry identified five proteins that stably interacted with budding yeast Rri1p/Csn5p in vivo at endogenous expression levels 28: (1.) the proteasome subunit Rpn5p/Nas5p 2930, (2.) Pci8p, a putative subunit of the translation initiation factor eIF3 31, and (3.) proteins encoded by the previously uncharacterized open reading frames YDR179C, YMR025W, and YOL117W (recently named RRI226). Specific interaction of these proteins in a complex is further confirmed by previous data from systematic two-hybrid screens 3233 (summarized in Fig. 1B). Pci8p and two of the three novel proteins contain PCI domains and show limited homology to fission yeast CSN subunits in various regions (Fig. 1A and data not shown). As the low degree of homology over the entire length of the proteins renders their assignment to individual metazoan CSN1-8 subunits uncertain, we refer to the previously uncharacterized genes according to a nomenclature proposed by Glickman and colleagues (personal communication). Previously assigned gene names were maintained, but supplemented by the names suggested by Glickman and colleagues, in order to indicate their common function in the budding yeast CSN complex, as documented in this report. The nomenclature used here is summarized in Fig. 1A.

As control of cullin deneddylation is one of the prominent activities of CSN 671925, we tested whether Rri1p/Csn5p-associated proteins regulate the modification state of the cullin Cdc53p with the budding yeast Ned8p homologue, Rub1p. Strains individually deleted for RRI1/CSN5, CSN9, RRI2/CSN10, PCI8/CSN11, and CSI1 (referred to as csn mutant strains throughout this manuscript) were obtained and Cdc53p modification was examined by immunoblotting of total cell lysate. Wild-type cells displayed two forms of Cdc53p migrating on SDS gels with different mobilities (Fig. 1C). Although the ratio of the two forms varied somewhat between individual experiments presumably due to slight variations in extract preparation, the faster migrating form represents unmodified Cdc53p, whereas the slower migrating form arises from Rub1p modification, as it was abolished in Δrub1 cells (Ref. 34 and Fig. 2A). Remarkably all five csn mutant strains accumulated Cdc53p exclusively in the Rub1p-modified form (Fig. 1C). Since deletion of RPN5/NAS5, a gene encoding another protein interacting with Rri1p/Csn5p is lethal due to its essential function in the proteasome 29, its effect on Cdc53p modification was not examined.

To determine whether budding yeast csn mutants exhibited a biochemical defect in the removal of Rub1p from Cdc53p similar to fission yeast csn mutants, we tested whether the readdition of cell lysate containing wild-type budding yeast CSN could rescue the Rub1p removal defect of csn deficient cell lysate. Total protein lysate from csn⁺ and Δrri1/csn5 cells prepared in the absence of exogenous ATP was incubated at a ratio of 1:2 for up to 60 minutes at 30°C, and Cdc53p modification was assessed by immunoblotting. To exclude readdition of Rub1p to unmodified Cdc53p during the incubation, the csn⁺ lysate was prepared from rub1 deletion strains as a source of functional CSN proteins. As previously described 34, Δrub1 cell lysate contained unmodified Cdc53p and no change in Cdc53p modification was observed upon incubation of this lysate for up to 60 minutes (Fig. 2A lanes 1–6). Likewise, incubation of Δrri1/csn5 lysate alone did not result in removal of Rub1p from Cdc53p over the 60 minute time course of the experiment (Fig. 2A, lanes 7–12).

When csn⁺Δrub1 and Δrri1/csn5 lysates were denatured in SDS sample buffer prior to mixing, the expected ratio of 1:2 was observed for Cdc53p/Cdc53p-Rub1p (Fig. 2A, lane 13, time point 0 minutes). In contrast, when native lysates were co-incubated at 30°C, the ratio reversed within 5 minutes, indicating complementation of the Δrri1/csn5 Rub1p removal defect by a functional CSN present in Δrub1 lysate (Fig. 2B, lanes). Identical complementation by Δrub1 lysate was obtained with several other Δcsn mutants (data not shown). These data show that, similar to fission yeast csn mutants, budding yeast Δcsn strains have a biochemical defect in the removal of Rub1p from Cdc53p, suggesting that the Rri1p/Csn5p, Csn9p, Rri2p/Csn10p, Pci8p/Csn11p, and Csi1p complex represents a budding yeast equivalent of the CSN complex described in fission yeast, plants, and human cells.

To substantiate this conclusion, we tested whether human CSN could complement the Rub1p removal defect of csn mutant cell lysates. Human CSN highly purified from erythrocytes (Fig. 2C; 3) was added to cell lysate prepared from all five csn mutants, and Rub1p modification of Cdc53p was assessed by immunoblotting. Addition of human CSN to csn deficient budding yeast cell lysate restored the ~1:1 ratio of modified to unmodified Cdc53p observed in wild-type cells, indicating efficient removal of Rub1p from Cdc53p in vitro (Fig. 2B). These results strengthen our conclusion that budding yeast csn mutants are deficient in a biochemical activity equivalent to human CSN.

While all five CSN proteins examined here are clearly involved in CSN-dependent cullin deneddylation, it is presently unclear whether they represent all subunits of the budding yeast CSN, in particular as all other known CSN complexes contain a minimum of eight subunits. It is equally unclear whether all five CSN proteins characterized here are part of the same complex. Although the budding yeast genome does not encode clearly discernible MPN or PCI domain proteins other than proteasome lid subunits and the CSN proteins characterized here (data not shown), it is possible that some of them dimerize to assemble a larger CSN complex. Alternatively, MPN and PCI domain containing proteasome lid subunits may substitute for authentic CSN subunits in budding yeast, thus giving rise to an eight-subunit complex. In support of this notion, Rpn5p/Nas5p, one of the non ATPase components of the 19S lid carrying a PCI domain that shares high homology with CSN4, was also found in the Rri1p/Csn5p complex isolated by tandem affinity purification 28. Similarly, Pci8p/Csn11p was previously shown to associate with eIF3 in budding yeast 31, suggesting a potentially complex pattern of CSN/eIF3/proteasome interactions. Further biochemical studies will be required to unravel this complexity and to reveal the exact stochiometry of the budding yeast CSN.

Studies in fission yeast implicated the PCI containing Csn1p and Csn2p, but not other CSN subunits 67, in a pathway required for the normal response to DNA damage and proper execution of S phase 5. These defects lead to cell elongation, slow growth, and sensitivity to UV, but not hydroxyurea (HU) 5. To examine whether these phenotypes are shared by budding yeast, all csn mutants were exposed to increasing doses of UV, the alkylating agent methyl-methane sulfonate (MMS), and HU. As shown in Fig. 3A,3B,3C, none of the budding yeast csn mutants were sensitive to either treatment. In addition, flow cytometric measurement of the DNA content in csn mutants did not show the accumulation of S phase cells characteristic of fission yeast csn1 and csn2 mutants 5 (Fig. 3D). These results indicate that the S phase and DNA damage response functions of CSN, unlike the deneddylation function, are not conserved among both yeasts. Since budding yeast lacks clearly identifiable homologues of csn1 and csn2, apart from their conserved PCI domains, it is possible that these proteins evolved specific domains required for these functions in fission yeast.

Our previous studies in fission yeast and the present studies in budding yeast showed that, apart from the S phase and DNA damage defects of fission yeast csn1 and csn2 mutants, the cellular phenotypes of yeast csn mutants are surprisingly unremarkable. In fact, no other gross growth or morphological phenotypes have been detected, despite the presence of maximally neddylated cullins in all yeast csn mutants examined (data not shown). Control of cullin deneddylation is therefore the only generally conserved biochemical function of CSN identified to date. In contrast, CSN has major developmental functions in multicellular eukaryotes, including D. melanogaster 35 and A. thaliana (reviewed in 36), although it is presently unclear whether any of these are linked to CSN-mediated deneddylation. While no substrates of the Rub1p/Ned8p modification system other than cullins have been identified, fission yeast csn1 mutants expressing tagged Ned8p accumulate several other proteins reactive with anti-tag antibodies 19. No major cellular pathway of unicellular organisms seems to be disturbed by hyperneddylation of these substrates, but their existence extends the biological functions of CSN to a potentially wider array of cellular processes. The unexpected conservation of the budding yeast CSN revealed in this report will offer novel opportunities for deciphering these functions in a simple, genetically and biochemically tractable organism.