Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

The microtubule organizing center regulates cytoskeletal integrity, and is termed the centrosome in higher eukaryotes, and the spindle pole-body (SPB) in yeast. The duplication of the SPB/centrosome is a key event that initiates entry into the cell cycle. The SPB is a proteinaceous structure that is embedded in the nuclear envelope, and its duplication marks exit from the G1 phase and the onset of chromosome duplication. The composition of the SPB has been defined, and the steps that lead to its duplication have been studied extensively using biochemical, genetic, and microscopic methods (3, 5, 6, 18, 24, 28, 30, 43, 44). An important component of the SPB is Cdc31 (3, 13), which in yeast is encoded by the essential CDC31 gene. CDC31 was isolated as a suppressor of kar1, a mutant that is unable to duplicate the SPB at the nonpermissive temperature (6). As with kar1, defects in CDC31 (cdc31) also cause G1 phase arrest, due to a failure in SPB duplication (48). Intriguingly, the kar1 defect was suppressed by Dsk2 (5), which has functional and structural resemblance to Rad23 (15, 29, 34, 40). The three centrin proteins that are expressed in human cells (30, 31) display differential tissue-specific levels (27, 36, 49). However, all three isoforms share the key structural features that are present in EF-hand proteins (32). Human CEN3 is closely related to Cdc31 and can interfere with SPB duplication when overexpressed in yeast (30). An N-terminal regulatory domain is proposed to regulate the activity of the C-terminal domain, possibly through Ca2+-mediated signaling (42). The C terminus binds various cellular proteins (1, 7, 11, 12, 16, 19-23, 28, 37, 46, 50), although the effect of these interactions in regulating growth control is not well understood. We found that the C-terminal domain of Cdc31 binds Rad4, and this interaction does not require Rad23. However, our findings indicate that Cdc31 forms regulated interactions with a preexisting complex comprising Rad23 and Rad4 that varies under different growth conditions. A mutant form of Cdc31, which formed a weak interaction with Rad4, showed reduced tolerance to UV-induced DNA damage. We report for the first time that centrin/Cdc31 plays an important role in protein degradation by the ubiquitin/proteasome system. Specifically, we discovered that Cdc31 binds the proteasome and can be purified with multiubiquitinated proteins. Moreover, mutant Cdc31 proteins had altered interactions with both proteasomes and ubiquitinated substrates and conferred proteolytic defects. A role for Cdc31 in protein degradation was implied by its interactions with Dsk2 (5), a protein that functions in the ubiquitin/proteasome pathway (15, 39, 40). We showed that a related protein, Rad23, is a shuttle factor that can translocate ubiquitinated proteins to the proteasome (8). The loss of both shuttle factors (rad23Δ dsk2Δ) causes a temperature-sensitive growth defect resulting from a failure to duplicate the SPB (5). Strikingly, high-copy-number expression of Cdc31 could overcome the SPB duplication defect of rad23Δ dsk2Δ (23) and predicted a role for protein degradation in regulating entry into the cell cycle. The presence of a single essential gene in Saccharomyces cerevisiae that encodes a centrin protein provides a genetically tractable system for characterizing the link between the cellular responses to stress and damage and the signaling pathways that control growth progression. For instance, DNA damage-induced growth arrest could be facilitated by blocking SPB duplication (1) in a mechanism involving the protein degradation function of Cdc31.