Disulfide Bond Formation in the ER
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.
Fidelity of Cargo Sorting
Vesicles that carry proteins from the ER to the Golgi are encapsulated by a set of coat proteins known as COPII. Two different processes, known as quality control mechanisms, select the correct subset of ER proteins to be admitted into COPII vesicles. The first process retains incompletely folded proteins within the ER by an unknown mechanism. We are currently devising genetic screens to identify mutations that allow inappropriate transport of incompletely folded proteins for genes required for proper protein retention. The second process involves targeting signals that allow the COPII coat proteins to selectively incorporate signal-bearing proteins into budding transport vesicles. The plasma membrane proton-ATPase (Pma1p) is one of the most abundant yeast membrane proteins and requires a number of specialized proteins for its correct exit from the ER. We identified, and are studying a gene called EXP1, which encodes a small membrane protein required for efficient transport of Pma1p. Exp1p cycles between the ER and Golgi and binds to the COPII subunit Sec24p, indicating that Exp1p acts as an adaptor for efficient packaging of Pma1p into COPII vesicle buds.
Regulated Sorting in the Late Secretory Pathway
In animal cells, some of the most interesting functions of the secretory pathway involve the regulated delivery of particular proteins to the cell surface in response to an environmental signal. We have discovered an analogous regulated secretory process in yeast, in which the integral membrane protein the general amino acid permease (Gap1p) is sorted in the trans-Golgi and endosome in response to the nitrogen source in the growth medium. We have identified a large collection of genes that are required for the proper sorting of Gap1p. These genes have revealed at least two stages for the proper intracellular sorting of Gap1p. In the first stage Gap1p is covalently modified by ubiquitination. The added ubiquitin tag acts to signal Gap1p trafficking to the vacuole; without ubiquitination all of the Gap1p is transported to the plasma membrane. In the second stage Gap1p can either be recycled to the Golgi (this step appears to be regulated by the abundance of amino acids) or Gap1p can enter luminal vesicles of the multivesicular endosome to ultimately be delivered to the interior of the vacuole.
Among the most interesting genes that govern Gap1p sorting are two small GTPases known as GTR1 and GTR2. These proteins, along with three additional polypeptides form a complex that is localized to the cytosolic face of the endosome. Mutations in any one of these genes prevents Gap1p cycling out of the endosome and causes constitutive delivery to the vacuole. Gtr2p can bind to the C-terminal tail of Gap1p and mutations in the tail that prevent Gtr2p binding will cause missorting of Gap1p to the vacuole. Taken together these results indicate that the Gtr proteins form a complex that is either part of a vesicle coat or is responsible for sorting Gap1p into recycling vesicles. We call this the GSE (GTPase containg complex for sorting in the endosome) and we are currently studying its structure and assembly on the membrane.
Many of the mutants that we identified that influence the intracellular sorting of Gap1p do so because they alter the intracellular abundance of amino acids. These mutants have thus provided access to the regulatory networks that respond to the availability of nutrients and control intracellular amino acid abundance by negative feedback. We are currently dissecting all of the different ways that nitrogen-derived signals are generated and how the membrane trafficking machinery responsible for sorting Gap1 decodes these signals.
Chen, E and C. A. Kaiser, LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J Cell Biol. 161:333-347 (2003).
Gao, M. and C.A. Kaiser, A conserved GTPase-containing complex is required for intracellular sorting of the general amino acid permease in yeast. Nat Cell Biol. 8:657-667 (2006).
Gross, E., C. Sevier, A. Vala, C.A. Kaiser and D. Fass, A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p. Nature Struct. Biol. 9:61-67 (2002).
Gross E, D. B. Kastner, C. A. Kaiser and D Fass, Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell. Cell 117:601-610 (2004).
Helliwell, S.B., S. Losko, and C. A. Kaiser, Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J. Cell Biol. 153: 649-662 (2001).
Kim, S., D.P. Sideris, C.S. Sevier, and C.A. Kaiser, Balanced Ero1 activation and inactivation establishes ER redox homeostasis. J Cell Biol. 196:713-25 (2012).
Risinger, A.L., N.E. Cain, E.J. Chen, and C. A. Kaiser, Activity-dependent reversible inactivation of the general amino acid permease. Mol Biol Cell. 17:4411-4419 (2006).
Sevier, C.S., H. Qu, N. Heldman, D. Fass, and C.A. Kaiser, Modulation of Cellular Disulfide Bond Formation and the ER Redox Environment by Feedback Regulation of Ero1. Cell 129, 333-344 (2007).