Hydroxyurea modulates thiol-disulfide homeostasis in the yeast endoplasmic reticulum

Hydroxyurea (HU) has been extensively used in laboratory settings to induce S-phase arrest and checkpoint activation. Furthermore, it has a history of clinical use as a cost-effective chemotherapeutic agent. Nevertheless, there is still uncertainty regarding its precise pharmacology, side effects, and toxicity, particularly in terms of its impact on organelle homeostasis. Here, we demonstrate that in budding yeast, HU specifically inhibits the endoplasmic reticulum-associated degradation (ERAD) of luminal misfolded proteins (ERAD-L pathway), an effect that is independent of S-phase arrest. In contrast, HU did not affect the degradation of misfolded ER membrane proteins or the degradation of cytosolic proteins. The selective inhibition of ERAD-L by HU is likely attributable to the formation of disulfide bonds in cysteine residues in luminal substrates, which must be reduced before their retrotranslocation to the cytosol. We further demonstrate that HU plays a role in alleviating reductive stress phenotypes observed in cells lacking Ero1, which is essential for oxidative protein folding in the ER. We propose that HU functions as a modulator of thiol-disulfide homeostasis in the ER lumen.