Genetic Requirements for Protein Degradation at the Endoplasmic Reticulum Translocon

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PROJECT SUMMARY Most endomembrane system and secreted eukaryotic proteins traverse the endoplasmic reticulum (ER) translocon during or shortly after synthesis. Underscoring the critical nature of maintaining functional translocons, eukaryotes possess multiple conserved translocon quality control (TQC) mechanisms that promote degradation of channel-clogging proteins. TQC remains poorly understood relative to other ER protein quality control pathways. The highly conserved yeast zinc metalloprotease Ste24 (ZMPSte24 in mammals) catalyzes degradation of translocon-clogging proteins. Preliminary data indicate that ER rhomboid-family pseudoprotease Dfm1 (homologous to mammalian derlin proteins) contributes to TQC, likely via the Ste24 mechanism. Consistent with the mission of the National Institute of General Medical Sciences, the objective of the proposed work is an improved understanding of TQC, a medically important facet of cell biology, using yeast as a model system. Ste24 promotes healthy aging and, by virtue of its function in TQC, is likely to play a protective role against the progression of diabetes. Further, at least two proteins with profound significance for metabolic physiology persistently engage translocons. The proposed experiments will test the hypothesis that Dfm1 and its cofactor, the Cdc48 ATPase, partially extract ubiquitylated clogging proteins from the translocon to enable cleavage by Ste24, whereupon the cytosolic fragment is degraded by the proteasome and the luminal fragment is trafficked through the endomembrane system to the vacuole for degradation. The specific aims of this project are to (1) characterize STE24 function in TQC and (2) determine the role of Dfm1 and Cdc48 in TQC. To address these objectives, several mechanistic aspects of Ste24 and Dfm1/Cdc48 function in TQC will be explored. The breadth of Ste24 and Dfm1/Cdc48 TQC substrates will be determined. Experiments will address whether Ste24 TQC substrates are ubiquitylated prior to recognition, if TQC substrates are cleaved in a Ste24-dependent manner, and how Ste24 TQC substrates are ultimately degraded (i.e. by the proteasome or vacuole). Dfm1 association with the translocon and TQC substrates will be assessed, as will the contributions of Dfm1 and Cdc48 to Ste24-mediated and Ste24-independent TQC. The role of conserved Dfm1 elements in TQC will be determined. Finally, yeast lacking DFM1 expressing TQC substrates exhibit a profound fitness defect that is rapidly and stably suppressed. The genetic determinants of suppression will be identified. Undergraduate and master’s students will participate in all aspects of this project, including experiment design, data collection, and communication of results. These experiments will yield novel mechanistic insights into conserved, biomedically relevant mechanisms of TQC. Upon completion, the proposed work has the strong potential to inform the development of improved therapeutic strategies for multiple human conditions, including elevated cholesterol and diabetes.