Abstract
Aims: Efficient oxidative protein folding (OPF) in the endoplasmic reticulum (ER) is a key requirement of the eukaryotic secretory pathway. In particular, protein folding linked to the formation of disulfide bonds, an activity dependent on the enzyme protein disulfide isomerase (PDI), is crucial. For the de novo formation of disulfide bonds, reduced PDI must be reoxidized by an ER-located oxidase (ERO1). Despite some knowledge of this pathway, the kinetic parameters with which these components act and the importance of specific parameters, such as PDI reoxidation by Ero1, for the overall performance of OPF in vivo remain poorly understood.Results: We established an in vitro system using purified yeast (Saccharomyces cerevisiae) PDI (Pdi1p) and ERO1 (Ero1p) to investigate OPF. This necessitated the development of a novel reduction/oxidation processing strategy to generate homogenously oxidized recombinant yeast Ero1p. This new methodology enabled the quantitative assessment of the interaction of Pdi1p and Ero1p in vitro by measuring oxygen consumption and reoxidation of reduced RNase A. The resulting quantitative data were then used to generate a simple model that can describe the oxidizing capacity of Pdi1p and Ero1p in vitro and predict the in vivo effect of modulation of the levels of these proteins.Innovation: We describe a model that can be used to explore the OPF pathway and its control in a quantitative way.Conclusion: Our study informs and provides new insights into how OPF works at a molecular level and provides a platform for the design of more efficient heterologous protein expression systems in yeast.
Highlights
The oxidative protein folding (OPF) pathway of the endoplasmic reticulum (ER) has been well characterized in the yeast Saccharomyces cerevisiae, both in terms of identification of components and defining how they interact
To trapping: Lane 1, post-Ni-NTA purification; lane 2, reduction of the Ni-NTA eluent by DTT (30 mM); lanes 3–6, reoxidation in the presence of flavin adenine dinucleotide (FAD) at 10 min, 30 min, 60 min, and 18 h; lane 7, purified Ero1p trapped with AMS; lane 8, purified Ero1p trapped with NEM. (C) Schematic showing the Ero1p activity assay where Ero1p reduced by DTT is reoxidized by O2, which is measurable using a Clarke electrode. (D) O2 consumption assay using a Clarke electrode
This yeast species has been successfully engineered to yield commercially viable levels, but this has been done empirically rather than in a directed and informed manner. The latter requires a systematic understanding of the yeast OPF pathway and its regulation using both in vivo and in vitro approaches, with the latter being hampered by inefficient methods for isolating key OPF components, in particular the ER-located oxidase Ero1p [57]
Summary
The oxidative protein folding (OPF) pathway of the endoplasmic reticulum (ER) has been well characterized in the yeast Saccharomyces cerevisiae, both in terms of identification of components and defining how they interact. The key components of the pathway are protein disulfide isomerase in yeast (Pdi1p) [13] and ER oxidase in yeast (Ero1p) [16, 41], encoded by the essential PDI1 and ERO1 genes, respectively. Groups of reduced proteins flow via Pdi1p to Ero1p through a series of thiol:disulfide oxidoreductions, to a bound flavin adenine dinucleotide (FAD) group within Ero1p, and thence to molecular oxygen (O2). There are marked differences between species in the number of members of the protein disulfide isomerase a Dave M.
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