Abstract
Srp1 (importin-α) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-α mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.
Highlights
Srp1 imports proteins containing nuclear localization signals (NLS) into the nucleus
We examined srp1-E402Q and srp1-55 mutants, which harbor distinct defects in nuclear import [20]. srp1-E402Q shows defective binding to a bipartite nuclear localization signal, and srp1-55 is unable to efficiently release NLS-containing substrates in the nucleus [20]
Importin-␣ is encoded by SRP1, and its role in NLSmediated protein import has been characterized extensively [20, 38, 39]
Summary
Srp imports proteins containing nuclear localization signals (NLS) into the nucleus. We determined that srp mutants that failed to import NLS-containing proteins (srp and srp1-55) successfully localized proteasomes to the nucleus. Srp mutants that did not target proteasomes to the nucleus (srp and srp1-E402Q) were able to import NLS-containing proteins. Co-expression of a member of each class of srp mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins. Sts did not suppress the nuclear import defect of srp, demonstrating that it contributes to the proteasome targeting role of Srp. These findings indicate that proteasome trafficking by Srp occurs by a mechanism that is distinct from its well characterized role in the import of nuclear proteins
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