Hydroxyurea inhibits ERAD-L independently of S-phase arrest in budding yeast

Abstract

Misfolded luminal and membrane proteins in the endoplasmic reticulum (ER) are recognized and retrotranslocated to the cytosol for proteasomal degradation, a process referred to as ER-associated degradation (ERAD). In Saccharomyces cerevisiae, ERAD substrates with luminal lesions are targeted for proteasomal degradation by the Hrd1 ubiquitin ligase complex (ERAD-L pathway). Membrane proteins containing lesions within their membrane-spanning regions are also targeted for degradation by the Hrd1 complex (ERAD-M pathway), while those containing lesions within their cytosolic regions are targeted for degradation mainly by the Doa10 ubiquitin ligase complex (ERAD-C pathway). Here, we demonstrate that hydroxyurea (HU), which is widely used to arrest cells in S-phase and is also used to manage several diseases including sickle cell anemia and chronic myeloproliferative disorders, inhibited ERAD-L, but not ERAD-M or -C. HU-mediated inhibition of ERAD-L occurred independently of S-phase arrest. In HU-treated cells, the integrity of the Hrd1 ubiquitin ligase complex remained intact and substrate recognition was unaffected. Moreover, induction of the unfolded protein response was undetectable in cells in which ERAD-L was inhibited by HU. These results suggest an unexpected action of HU, which modulates protein quality control in the secretory pathway, and also suggest the existence of an additional regulatory step in ERAD.