The Discovery of Molecules and Mechanisms of the Rhomboid Superfamily

Project Summary/Abstract Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptably low levels. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD substrates are selectively extracted from the ER before degradation by the cytosolic 26S proteasome. Despite its commonality in ERAD, many features of retrotranslocation have remained mysterious. We have made a major breakthrough in understanding retrotranslocation. By employing whole-genome yeast arrays, we have discovered the rhomboid family protein Dfm1 to be critical for the removal of membrane substrates and has opened the door to a deep mechanistic understanding of retrotranslocation and rhomboid protein biology. Notably, we identified two additional biological functions of derlin Dfm1 where 1) it employs a chaperone-like role for alleviating aggregated misfolded membrane protein stress and 2) regulates sphingolipid metabolism. Overall, this places Dfm1 at the heart of vast membrane-related processes. To gain deeper biological insights from these new findings, we will: 1) Define the mechanism associated with Dfm1-mediated retrotranslocation. 2) Explore a new stress-state associated with aggregated misfolded membrane proteins. 3) Determine how Dfm1 regulates sphingolipid metabolism. We will use a multifaceted approach including biochemistry, cell biology, genetics, structural biology, proteomics, and advanced microscopy to address these central questions in rhomboid proteins and membrane biology. We will leverage our unique in vivo and in vitro assays-and continue to devise new ones-to dissect the basic mechanisms of rhomboid-mediated retrotranslocation and its place in cell and organismal biology. A mechanistic understanding of rhomboid protein biology in protein quality control, stress alleviation, and lipid metabolism will establish foundational biological insights while unveiling therapeutic targets for a variety of critical pathways including protein misfolding, protein quality control, ER stress, and lipid dysregulation.