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Quantitative Analyses of the Yeast Oxidative Protein Folding Pathway in Vitro and in Vivo

Beal, David M, Bastow, Emma L., Staniforth, Gemma L., von der Haar, Tobias, Freedman, Robert B., Tuite, Mick F. (2019) Quantitative Analyses of the Yeast Oxidative Protein Folding Pathway in Vitro and in Vivo. Antioxidants & Redox Signalling, 31 (4). pp. 261-274. ISSN 1523-0864. (doi:10.1089/ars.2018.7615) (KAR id:73366)

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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 disulphide bonds, reduced PDI must be re-oxidised 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 oxidised 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 RNAase A. The resulting quantitative data were then used to generate a simple model which can describe the oxidising capacity of Pdi1p and Ero1p in vitro and predict the in vivo effect of modulation of the levels of these proteins.

Conclusion: Our study 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.

Item Type: Article
DOI/Identification number: 10.1089/ars.2018.7615
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Susan Davies
Date Deposited: 04 Apr 2019 08:58 UTC
Last Modified: 06 May 2020 03:19 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/73366 (The current URI for this page, for reference purposes)
Beal, David M: https://orcid.org/0000-0002-1819-315X
von der Haar, Tobias: https://orcid.org/0000-0002-6031-9254
Tuite, Mick F.: https://orcid.org/0000-0002-5214-540X
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