Biochemical basis of oxidative protein folding in the endoplasmic reticulum

This work has focused on understanding how disulfide bonds are formed in folding proteins as they travel through the secretory pathway. The endoplasmic reticulum (ER) supports the formation of disulfide bonds in newly translocated proteins through a mechanism dependent on the protein Ero1p (ER oxidoreductin 1). I developed a purification procedure to purify Ero1p from yeast microsomes and determined that it bound flavin adenine dinucleotide (FAD). With this finding, I was able to reconstitute efficient oxidative folding in vitro using purified Ero1p, protein disulfide isomerase (PDI), and FAD. I demonstrated that Ero1p is a specific, FAD-dependent oxidase of PDI, which subsequently introduces disulfide bonds into folding proteins. The catalysis of disulfide formation by Ero1p and PDI proceeds through a series of disulfide exchange reactions and is not dependent on glutathione. FAD-bound Ero1p then uses molecular oxygen as its terminal electron acceptor to reoxidize itself. In parallel, by depleting various redox molecules from yeast and examining the consequences on disulfide formation, we were able to confirm that oxidative folding in vivo is highly dependent on cellular levels of FAD.; Ero1p-catalyzed disulfide formation proceeds rapidly even in the presence of reduced glutathione. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfides. Glutathione is not the primary source of oxidizing equivalents, but functions instead as a net reductant. The high oxidized glutathione content in the ER is an indirect consequence of Ero1p activity.; Ero1p is the first described flavoprotein localized entirely within the ER, defining a novel role for FAD within the ER lumen. I have also established the presence of a robust transport system that imports FAD into the ER. The Ero1p oxidation system is highly sensitive to physiological levels of free FAD. High levels of free FAD stimulate the Ero1p-driven oxidation cycle, while low levels hinder the cycle. Overall, I have determined the biochemical basis of oxidative protein folding in the endoplasmic reticulum, and my results suggest that controlling the levels of FAD available to Ero1p could be a means to regulate oxidative folding according to a cell's nutritional or metabolic state.