Cdc31 and Dsk2 were isolated as suppressors of a kar1
mutant, which is defective in SPB duplication. Dsk2 resembles Rad23, since both proteins contain proteasome-binding (UBL) and ubiquitin-binding (UBA) domains. Rad23 functions as a shuttle factor that can deliver multiubiquitinated substrates to the proteasome, and a similar function is predicted for Dsk2, which can also bind multiubiquitinated proteins and the proteasome. Furthermore, Dsk2 has partially overlapping roles with Rad23 (39
), and both proteins have biochemical and genetic interactions with Cdc31. The association of Cdc31 with two related proteolytic factors anticipated a role for protein degradation in SPB duplication and cell cycle control. It is significant in this regard that loss of both Dsk2 and Rad23 (dsk2
Δ) causes a failure in SPB duplication that could be suppressed by Cdc31. A role for Rad23 in cell cycle control was suggested by the defective SPB duplication in a rad23
Δ mutant (5
) and by a transient G2
phase growth delay in a rad23
Δ mutant (26
). Taken together, these diverse genetic and biochemical findings suggest that Cdc31, Rad23, and Dsk2 function in a common pathway that regulates cell cycle progression.
The regulation of Cdc31 function is unclear although in metazoans Ca2+
is likely to be a critical signaling mediator. Because yeast Cdc31 interacts very weakly with Ca2+
), it is uncertain if its interactions with multiubiquitinated proteins and proteasomes are regulated by this or by other divalent metals. In addition to Ca2+
-binding, a poorly described covalent modification of human centrin has been described (36
). Our characterization of FLAG-Cdc31 by 2D gel electrophoresis also provides evidence for extensive modification. However, further study will be required to determine if the posttranslational modification of Cdc31 affects its interaction with cellular proteins. For instance, it would be interesting to determine if Cdc31 forms exclusive interactions with Rad4 and the proteasome and if these interactions are regulated following DNA damage.
Centrins are conserved EF-hand proteins bearing strong structural similarity to the calmodulin family of regulatory proteins (4
). The yeast Cdc31 protein contains two pairs of EF hands that are separated by a linker sequence (Fig. ). Each pair operates as a functional unit, although only the carboxyl pair of EF hands in yeast Cdc31 has been reported to bind calcium (37
). Most, if not all, Cdc31/centrin interactions are mediated by the carboxy-terminal domain. However, the EF hands in the amino-terminal domain may influence the function of the C terminus. Cdc31 has been reported to bind SPB components (5
), regulators of mRNA export (14
), DNA repair factors (1
), and signal transduction proteins (23
). The Kar1 and Dsk2 proteins play an important role in targeting Cdc31 to the SPB. Cdc31 binds Sfi1, a central component in the SPB (28
). An intriguing question is how the centrin proteins bind such a diverse collection of proteins through a single domain. An important clue to the nature of this interaction is offered by the characterization of calmodulin, which binds an amphiphilic α-helix in its target proteins. Similar structures have been identified in Kar1 and XPC/Rad4 (7
). The interactions between Cdc31 and diverse cellular proteins were revealed in genetic studies (23
) and are consistent with multiple roles. Moreover, a significant fraction of Cdc31 is not present in the SPB (36
). In addition to SPB duplication defects, cdc31
mutants have also shown cell morphology and integrity defects (45
Human CEN2 was discovered in a complex with the DNA repair factors XPC and hHR23 (1
) and was reported to stimulate XPC-mediated DNA incision (33
). The related DNA repair proteins in yeast are encoded by the RAD4
genes, and both proteins are present in the NEF2 repair complex (17
). The interaction between Cdc31 and NEF2 could provide a way to convey the cellular response to DNA damage to the pathways that arrest cell cycle progression. To explore these ideas we characterized the single centrin-encoding gene in yeast (CDC31
). We show here that yeast NEF2 contains Cdc31. Although this interaction is mediated by Rad4 and does not require Rad23, we note that Cdc31 forms regulated interactions with a preassembled Rad23/Rad4 complex. Significantly, a cdc31
mutant that formed reduced binding to Rad4 showed sensitivity to UV-induced DNA damage, suggesting that Cdc31 might promote the cellular response to DNA damage by regulating cell cycle progression.
We describe significant new interactions that define a novel role for centrin/Cdc31 in the ubiquitin/proteasome system. Cdc31 can bind the 26S proteasome. Cdc31 can also be copurified with multiubiquitinated proteins, which could reflect its interaction with the proteasome. Additionally, Cdc31 is conjugated to one to three ubiquitins, although it is a stable protein. Since the presence of one to three ubiquitins does not promote a stable interaction with the proteasome, we speculate that this modification might affect Cdc31 function. We note, for instance, that oligomeric forms of ubiquitinated Cdc31 were not detected (Fig. ), suggesting that ubiquitination might prevent self-assembly and aggregation.
We found that Cdc31 could bind several AAA class ATPases that are present in the 19S regulatory particle. Although the effect of this interaction is currently not known, we stress that this binding with purified proteins strongly validates our hypothesis that Cdc31 can interact with the proteasome. Furthermore, these in vitro results demonstrate that the Cdc31-proteasome interaction does not require prior attachment to a multiubiquitin chain.
We propose that Cdc31 interactions with the ubiquitin/proteasome system are biologically significant because cdc31 mutants are highly sensitive to drugs that generate protein damage and are unable to degrade proteolytic substrates efficiently. Moreover, the cdc31-2 protein interacted at dramatically reduced levels with both proteasomes and multiubiquitinated proteins.
The binding of Cdc31 to its various cellular partners (including XPC/Rad4, Kar1, Sfi1, proteasome, and multiubiquitinated substrates) is mediated by its carboxy-terminal domain. Removal of the amino terminus resulted in ~20-fold decreased abundance of the carboxy-terminal domain (cdc31-C), although a strong interaction with its cellular partners was retained. In contrast, the amino-terminal domain (cdc31-N) was stable but did not bind any of the aforementioned proteins. These results are consistent with the view that the amino-terminal EF hand exerts a regulatory effect. Moreover, both domains are essential for function since neither cdc31-N nor cdc31-C could suppress the inviability of cdc31Δ.
The regulation of Cdc31 activities is not well understood. Although Cdc31 belongs to a family of conserved Ca2+
-binding proteins, it does not bind Ca2+
with high affinity. Cdc31 interactions with Kic1 and Kar1 (6
), as well as its binding to proteasomes and multiubiquitinated proteins, are apparently Ca2+
independent. Cdc31 interactions with the proteasome and multiubiquitinated proteins were similarly unaffected by EGTA or excess Ca2+
(data not shown). In contrast, studies using purified human CEN2 and the carboxy-terminal EF-hand domain showed Ca2+
-dependent interaction with a peptide derived from XPC (37
). Subtle changes in calcium levels may affect Cdc31 structure and influence its interaction with the proteasome and multiubiquitinated proteins.
CDC31 encodes the essential Cdc31 protein in S. cerevisiae.
The availability of genetic mutants permitted our preliminary characterization of Cdc31 and our discovery that it plays an important role in protein degradation. Cdc31 mutants harboring defects in various cellular functions have been identified and characterized. Because of Cdc31's critical role in controlling SPB duplication, Cdc31 might be ideally positioned to regulate cell cycle arrest in response to environmental stresses. Therefore, we speculate that an important role for Cdc31 might involve integrating a DNA damage signal to execute checkpoint growth arrest. Delaying SPB duplication is expected to facilitate efficient NER. This model is consistent with the UV sensitivity of the cdc31-1 mutant, the previously described interaction between centrin and XPC, and our finding that Cdc31 binds NEF2. However, it remains to be determined if the proteolytic functions of Cdc31 are required for a putative checkpoint function. We note that a number of genetic and biochemical interactions with components of the ubiquitin/proteasome system (including Dsk2 and Rad23) have been described, and it is significant that double mutants involving Rad23 (rad23Δ rpn10Δ or rad23Δ dsk2Δ) cause defects in cell cycle control.