Principal Investigator Chris Kaiser
Most secreted proteins and extracellular domains of membrane proteins contain disulfide bonds. These covalent bonds are important for the proper folding and stability of secretory proteins, and experimentally they can be used as covalent probes of in vivo folding intermediates. It has long been known that disulfide bonds form as newly synthesized proteins enter the ER although the enzymatic pathway for the formation of disulfide bonds has only recently come to light. Our work on this problem began with the identification of the yeast gene, ERO1, which encodes a flavoprotein oxidase that is the primary source of disulfide bonds in the ER. Native disulfide bond also requires protein disulfide isomerase (PDI). PDI and Ero1p are linked by a disulfide relay in which disulfide bonds formed within Ero1p are transferred to PDI, which in turn transfers disulfide bonds to substrate proteins. Genetic screens have also revealed a minor pathway for ER oxidation that involves another flavoprotein known as Erv2p. Like Ero1p, Erv2p can transfer a disulfide bond to PDI as a cofactor.
We have undertaken a collaborative effort to understand the mechanism both Ero1p and Erv2p by structural analysis using X-ray crystallography. Although these two proteins do not share sequence similarity, their basis structures are quite similar. Both proteins contain an anti-parallel four-helix bundle that holds FAD in proximity to two cysteins that can form a disulfide at the active site. In addition both proteins contain a second pair of cysteines on a mobile peptide segment that can engage in disulfide exchange with the cysteines of the active site. We believe that this disulfide shuttle mechanism is crucial for the ability of both Ero1p and Erv2p to transfer disulfides specifically to PDI rather than to other free thiols in the ER such as glutathione. We are currently using biochemical and genetic methods to study the mechanisms that allow specific disulfide transfer reactions to take place.