In this study, we mapped the binding of the prototypical substrate-processing cofactors Ufd2 and Ufd3 to overlapping but nonidentical sites at the C terminus of Cdc48. We showed for the first time that the C-terminal tail of Cdc48 is both necessary and sufficient for Ufd2 binding and that it is the only binding site for Ufd2. Importantly, the mapping of the binding sites allowed us to generate cdc48 mutant yeast strains defective in Ufd2 and Ufd3 binding and to demonstrate that key cellular functions of Ufd2 and Ufd3 depend on their interaction with Cdc48.
Within the six C-terminal residues of Cdc48 mediating binding to Ufd2 and Ufd3, residue Y834 was found to be of particular importance. The homologous residue Y805 of p97 has previously been shown to be critical for binding of the structurally unrelated PUB and PUL domains to p97 (68
). We show here that Y834 is crucial for Ufd2 binding as well, identifying this residue as a key determinant for binding of all cofactors known to interact with the C terminus of Cdc48/p97. Importantly, however, our results indicate that Ufd3, in contrast to Ufd2, possesses additional binding determinants, in agreement with a recent study reporting that a p97-C10 peptide has a lower affinity for the PUL domain of PLAA than either p97-C13 or full-length p97 (67
). The higher affinity of the longer peptide suggests that critical contacts between the PUL domain and p97 may be missing in a recent crystal structure of the PUL domain from PLAA (50
). This structure was obtained with the p97-C10 peptide and revealed contacts between the PUL domain and residues L804 and Y805 of p97, whereas the five highly conserved acidic residues preceding L804 were disordered. It is tempting to speculate that residues upstream of the sequence covered by p97-C10 make additional contacts with the PUL domain, thus permitting electrostatic interactions between the highly conserved acidic p97 residues and a conserved positively charged groove of the PUL domain adjacent to the binding site for L804 and Y805.
Cell cycle-dependent phosphorylation of Cdc48 residue Y834 has been reported to control subcellular localization of Cdc48 and cell growth (41
). We failed to detect significant phosphorylation of Y834 using Cdc48 and phosphotyrosine antibodies that sensitively detected their respective antigens (d), suggesting that, at best, only a very minor subpopulation of Cdc48 is phosphorylated on Y834. Using strains that express mutated Cdc48 under the control of the CDC48
promoter at wild-type levels, we also failed to detect a slow-growth phenotype for the cdc48
mutant, in contrast to Madeo et al. (41
), and demonstrated normal cell cycle progression for all mutants tested (c). These results clearly indicate that either phosphorylation of Y834 is not required for the cell cycle-dependent localization of Cdc48 or cell cycle-dependent localization of Cdc48 is not essential for normal cell cycle progression. However, a potential physiological role of Y834 phosphorylation (if existent) in controlling cofactor binding cannot be formally excluded, in part because the binding stoichiometry between Cdc48 and Ufd2/Ufd3 in vivo
is unknown. We found that wild-type and Y834E protomers form mixed Cdc48 hexamers that still bind Ufd2 and Ufd3, proving that Ufd2/Ufd3 binding is not abolished by a single mutant protomer and suggesting that one or a few wild-type protomers per hexamer may be sufficient for Ufd2/Ufd3 binding (d). If a single unphosphorylated C-terminal binding site within a Cdc48 hexamer were indeed sufficient for a normal interaction with Ufd2 and Ufd3, then an efficient binding control would require the concerted phosphorylation of Y834 on all six subunits of the hexamer. This scenario is highly unlikely to represent a major mechanism for the control of Ufd2/Ufd3 binding, given our failure to detect significant levels of Y834 phosphorylation in unstressed, logarithmically growing yeast cells even upon enrichment of Cdc48 by immunoprecipitation (d). It should be noted though that our analysis of tyrosine phosphorylation in whole-cell extracts does not exclude the formal possibility that very small subpopulations of Cdc48 are phosphorylated on all six Y834 residues to control cofactor interactions in a spatially and/or temporally restricted manner. Such a regulatory mechanism would presumably rely on a pulse of tyrosine kinase activity, and phosphorylation of p97 residue Y805 is indeed induced in response to T cell activation (13
). The future identification of the kinase(s) catalyzing the phosphorylation of Cdc48 on residue Y834 (if existent) will be a prerequisite to further analyzing the significance of Y834 phosphorylation in yeast.
This study shows that cdc48
mutants altered in the Ufd2/Ufd3 binding site are impaired in the OLE, UFD, and ERAD pathways and in cellular stress responses, indicating that Cdc48 binding is required for well-known cellular functions of Ufd2 and Ufd3. Ufd2 is a prototypical E4 ubiquitin chain elongating enzyme and Cdc48 substrate-processing cofactor (35
). Nevertheless, its ability to directly interact with E2 ubiquitin-conjugating enzymes (46
) as well as with the ubiquitin binding protein Rad23 (24
) raised the possibility that Ufd2 may bind and multiubiquitylate preubiquitylated substrates independently of Cdc48. However, the phenotypes of the cdc48
mutants clearly indicate that Ufd2 cannot function independently of Cdc48 in the OLE and UFD protein degradation pathways (b to d) and suggest that all cellular functions of Ufd2 depend on Cdc48 binding. This would imply that all Ufd2 substrates are also Cdc48 substrates, a prediction supported by a recent study characterizing novel Ufd2 substrates (40
Our phenotypic analysis of canavanine sensitivities indicated that cdc48
C-terminal mutants are nearly as sensitive as the ufd3
mutant expressing Ufd3 mutated in residues R541 and R669 but less sensitive than the Δufd3
mutant (a). While these data might suggest that the defects leading to the hypersensitivity of the Δufd3
mutant are partially Cdc48 independent, the interpretation is complicated by the fact that phenotypes of Δufd3
are attenuated by deletion of UFD2
). Because the cdc48
C-terminal mutants are deficient in Ufd2 and Ufd3 binding, it is not possible to differentiate between the possibilities of additional Cdc48-independent defects in the Δufd3
mutant on one hand and attenuation caused by impaired Ufd2 binding in the cdc48
mutants on the other hand. A recent study employing a ufd3
strain mutated in Ufd3 residues I483, D538, and L571 came to the conclusion that Cdc48-independent functions of Ufd3 exist (50
). While more detailed structural insights into the interaction between Cdc48 and Ufd3 have to await a cocrystal structure analysis of both full-length proteins, the phenotypic analysis by Qiu et al. is difficult to reconcile, as the well-characterized canavanine hypersensitivity of ufd3
mutants (a) (39
) was not observed in their experimental system (50
). The authors also failed to demonstrate mutual loss of binding for the ufd3
mutants used, leaving the possibility that residual interactions between Cdc48 and Ufd3 caused the lack of some phenotypes. Thus, while we cannot formally exclude the existence of Cdc48-independent functions of Ufd3, unequivocal evidence supporting such a possibility is lacking.
Interestingly, the Y834F mutation caused only a partial loss of function in vivo
, whereas the phenotypes of all other cdc48
C-terminal mutants resembled those of Δufd2
mutants. A likely explanation is that the Y834F mutation does not completely abolish Cdc48 binding to Ufd2 and Ufd3. While atomic details of the interaction between Cdc48 and Ufd2 are not available, the interaction between the phenolic ring of residue Y834/Y805 with the PUB and PUL domains is mediated by both hydrogen bonds of the hydroxyl group and van der Waals interactions of the aromatic ring (50
). The latter may well be preserved in the Y834F mutant but not the other mutants, raising the possibility of a weak residual binding of Y834F to Ufd2 and Ufd3 that would be consistent with its milder phenotypes. Indeed, weak binding of Y834F suggestive of residual, transient interactions could be observed for Ufd3 in vitro
(e) and, albeit variably, for Ufd2 in some but not all immunoprecipitation experiments (data not shown).
Surprisingly, and in contrast to that of Ufd3, the Cdc48 binding site of Ufd2 is not conserved throughout evolution. In higher eukaryotes, binding relies on the interaction of a VBM in Ufd2/E4B with the N domain of Cdc48/p97 () (6
). Remarkably, E4B does not bind to the C-terminal tail of p97 (15
), even though the sequence of the tail is extremely conserved from yeast to humans. This suggests that an active counterselection against the (elusive) Cdc48 binding site of yeast Ufd2 must have existed during evolution. One can only speculate about potential evolutionary driving forces behind this unusual finding. However, it seems plausible that the transformation of fungal Ufd2 proteins into N domain interactors in higher eukaryotes was coupled to the emergence of the PUB domain, which binds to the C-terminal tail of Cdc48/p97 and is found in all eukaryotes but fungi (12
). Perhaps the benefits of a diversification of Cdc48/p97-cofactor complexes and their cellular functions gained from PUB domain proteins outweighed the costs of having to adapt to a new Ufd2 binding mechanism.