ENSURING THE INTEGRITY OF THE SECRETORY AND MEMBRANE PROTEOME
The ER is the "port of entry" for proteins destined for the cell surface and beyond. The vast majority of proteins entering the secretory pathway are synthesized on ribosomes docked at ER translocons and are co-transationally translocated into the ER lumen. Proteins synthesized at the ER are subject to covalent modifications that include N- and O-glycosylation, disulfide bond formation, and in some cases, proline and lysine hydroxylation. Membrane proteins must be threaded co-translocationally into the lipid bilayer to become membrane-integrated, often with complex topologies and typically form hetero- or homo- oligomers. This highly complex "protein biogenesis" process is assisted by a diverse network of folding catalysts and protein-modifying enzymes and is scrutinized by molecular chaperones and other "quality control" factors which ensure that only correctly folded and assembled proteins exit the ER and proceed to distal compartments of the secretory pathway.
Our goal is to elucidate the functional networks that coordinate protein synthesis and quality control in the early secretory pathway. Currently the lab is focused on two specific systems: ERAD and ribosome UFMylation.
ER-ASSOCIATED PROTEIN DEGRADATION (ERAD)
Folding begins during translocation and persists after the polypeptide chain has been released by the ribosome. Folding intermediates are not competent for export out of the ER and may undergo multiple rounds of folding attempts, with different proteins folding at different rates. Some proteins are unable to achieve a native conformation because of the presence of mutations, amino acid mis-incorporation, or unavailability of oligomeric partners. Triage refers to the process by which folding-defective proteins become committed to ERAD. Because protein degradation is mediated by the ubiquitin-proteasome system in the cytosol, in order to be degraded, proteins - or parts of proteins
that were translocated into the ER lumen - must be dislocated back across the ER bilayer, through a proteinaceous dislocon. This process requires metabolic energy and covalent modification by ubiquitin. Because ERAD clients are often very hydrophobic, the dislocation process is thought to be tightly coupled to proteolysis in order to avoid the release of aggregation-prone intermediates to the cytosol.
Accommodating structural and topological diversity in the ER
Proteins synthesized in the ER are incredibly diverse in terms of their transmembrane topologies, post-translational modifications, oligomeric and oxidation states. How the triage and ERAD machinery is able to deal with this diversity is not well understood. Our research seeks to understand the molecular mechanisms underlying substrate selectivity in ERAD. What are the "triage factors" that commit slow- folding proteins to ERAD? What features within the nascent chain are recognized? How does this machinery accommodate diversity among structures, topologies and folding rates?
Systems-level dissection of mammalian ERAD
To address these questions, in a recent study, we used genome-wide CRISPR/Cas9 analysis to construct a fine-grained map of the molecular determinants that underlie degradation of structurally and topologically diverse luminal, membrane and cytosolic ERAD clients, generating protein-specific genetic fingerprints and revealing the identity of distinct ERAD modules specialized for different substrate classes. Extending this work with single and double mutant screens is one strategy aimed at gaining a comprehensive understanding of the rules that govern protein triage and quality control in the early secretory pathway.
UFM1 is a ubiquitin-like protein modifier that shares with ubiquitin a common structural fold, analogous chemistry and enzymology. Like ubiquitin, UFM1 is covalently conjugated to proteins via an isopeptide linkage between its C-terminal Gly and amino groups on target protein lysines. We identified the entire UFM1 conjugation and de-conjugation pathway in a genome-wide screen for ERAD and identified the ribosomal protein RPL26 as the principal substrate of UFMylation. RPL26 is positioned adjacent to the polypeptide exit site of the ribosome and specifically UFMylated at two lysine residues on a helical extension of RPL26 that co-evolved with UFM1 and its conjugation system. Both UFMylation and de-UFMylation of RPL26 occur exclusively at the cytoplasmic surface of the ER membrane, and UFMylation is stimulated by conditions that cause ribosomes to stall and collide while co-translationally synthesizing secretory and membrane proteins at ER translocons. Current biochemical, genetic and structural studies are focused on elucidating how UFMylation promotes resolution of ER-stalled ribosomes.