Mechanisms of disulfide bond formation in the endoplasmic reticulum

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Proteins destined for the extracellular space fold and form disulfide bonds in the endoplasmic reticulum (ER) prior to further traversing the secretory pathway. Disulfide bonds can be introduced in a redox relay where electrons are passed from the client protein through a protein disulfide isomerase and an oxidase to O2. However, many lines of evidence support the existence of alternative pathways for disulfide bond formation. The focus of this research program is to identify novel pathways for disulfide bond formation in the ER. To this end we have taken an unbiased discovery approach to identify proteins that interact transiently with a canonical ER client, vascular endothelial growth factor A (VEGF-A), during its maturation in the ER. Using proximity labeling, isolation and mass spectrometry, we discovered a strong enrichment for mitochondrial proteins in the vicinity of VEGF-A. This suggests that VEGF-A matures in an ER sub-domain in close contact to the mitochondria called the mitochondria associated membrane (MAM), known to be rich in folding factors. This has led us to hypothesize that ER-mitochondrial contact sites (MERCs) support disulfide bond formation in the ER. Interestingly, the mitochondrial intermembrane space (IMS) is the only other cellular compartment that is capable of disulfide bond formation. Objective 1 of this project is to determine the importance of MERCs for ER localized protein folding. We will genetically disrupt the MERCs and measure the impact on maturation of VEGF-A and other ER clients, as well as overall secretion capacity. We will also monitor activation of the Unfolded Protein Response which reports on ER folding stress. Objective 2 is to identify mitochondrial processes that contribute to ER-localized protein folding. We will genetically or pharmacologically disrupt the pathway involved in disulfide bond formation in the IMS and monitor the impact on protein maturation and UPR as above. We will also determine if other mitochondrial processes such as the electron transport chain and the tricarboxylic acid cycle play roles in supporting ER-localized oxidative protein folding. Objective 3 is to determine the importance of MERCs for cell-cell interactions. We will apply conditioned media from cells with disrupted MERCs to other cell types including endothelial cells, macrophages and adipocytes. We will measure the ability of the secretome to stimulate cell physiological processes such as endothelial tube formation, polarization and glucose uptake. Disulfide bond formation is an essential process important for numerous cellular and organismal phenotypes. However, there are large gaps in our knowledge about the mechanisms involved in this process. This research program aims to identify novel pathways for disulfide bond formation and map a unique relationship between the function of the ER and mitochondria important for secretion-mediated phenotypes.