The Cellular Location of Rad23, a Polyubiquitin Chain-Binding Protein, Plays a Key Role in Its Interaction with Substrates of the Proteasome.

Well-studied structural motifs in Rad23 have been shown to bind polyubiquitin chains and the proteasome. These domains are predicted to enable Rad23 to transport polyubiquitylated (polyUb) substrates to the proteasome (Chen and Madura, 2002 [1]). The validation of this model, however, has been hindered by the lack of specific physiological substrates of Rad23. We report here that Rad23 can bind Ho-endonuclease (Ho-endo), a nuclear protein that initiates mating-type switching in Saccharomyces cerevisiae. We observed that the degradation of Ho-endo required export from the nucleus, in agreement with a previous report (Kaplun et al., 2003 [2]), and suggests that Rad23 can traffic proteins out of the nucleus. In agreement, the subcellular distribution of Rad23 is noticeably altered in genetic mutants that disrupt nucleocytoplasmic trafficking. Significantly, the location of Rad23 affected its binding to polyUb substrates. Mutations in nuclear export stabilized substrates, and caused accumulation in the nucleus. Importantly, Rad23 also accumulated in the nucleus in an export mutant, and bound to higher levels of polyUb proteins. In contrast, Rad23 is localized in the cytosol in rna1-1, a nucleocytoplasmic transport mutant, and it forms reduced binding to polyUb substrates. These and other studies indicate that substrates that are conjugated to polyubiquitin chains in the nucleus may rely on an export-dependent mechanism to be degraded by the proteasome. The evolutionary conservation of Rad23 and similar substrate-trafficking proteins predicts an important role for export in the turnover of nuclear proteins.

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