Browsing by Author "Rubenstein, Eric M."

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    Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans
    (PeerJ, 2023-08-25) Doss, Ellen M.; Moore, Joshua M.; Harman, Bryce H.; Doud, Emma H.; Rubenstein, Eric M.; Bernstein, Douglas A.; Biochemistry and Molecular Biology, School of Medicine
    Background: Candida albicans is the most prevalent human fungal pathogen. In immunocompromised individuals, C. albicans can cause serious systemic disease, and patients infected with drug-resistant isolates have few treatment options. The ubiquitin-proteasome system has not been thoroughly characterized in C. albicans. Research from other organisms has shown ubiquitination is important for protein quality control and regulated protein degradation at the endoplasmic reticulum (ER) via ER-associated protein degradation (ERAD). Methods: Here we perform the first characterization, to our knowledge, of ERAD in a human fungal pathogen. We generated functional knockouts of C. albicans genes encoding three proteins predicted to play roles in ERAD, the ubiquitin ligases Hrd1 and Doa10 and the ubiquitin-conjugating enzyme Ubc7. We assessed the fitness of each mutant in the presence of proteotoxic stress, and we used quantitative tandem mass tag mass spectrometry to characterize proteomic alterations in yeast lacking each gene. Results: Consistent with a role in protein quality control, yeast lacking proteins thought to contribute to ERAD displayed hypersensitivity to proteotoxic stress. Furthermore, each mutant displayed distinct proteomic profiles, revealing potential physiological ERAD substrates, co-factors, and compensatory stress response factors. Among candidate ERAD substrates are enzymes contributing to ergosterol synthesis, a known therapeutic vulnerability of C. albicans. Together, our results provide the first description of ERAD function in C. albicans, and, to our knowledge, any pathogenic fungus.
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    Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon
    (Caltech LIbrary, 2023-11-09) Runnebohm, Avery M.; Indovina, Christopher J.; Turk, Samantha M.; Bailey, Connor G.; Orchard, Cade J.; Wade, Lauren; Overton, Danielle L.; Snow, Brian J.; Rubenstein, Eric M.; Biochemistry and Molecular Biology, School of Medicine
    Proteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.