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Overexpression of Membrane Proteins in Saccharomyces cerevisiae for Structural and Functional Studies: A Focus on the Rabbit Ca2+-ATPase Serca1a and on the Yeast Lipid “Flippase” Complex Drs2p/Cdc50p

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Membrane Proteins Production for Structural Analysis

Abstract

Membrane proteins (MPs) such as transporters, receptors, and ion channels exert key cellular functions. MPs account for one quarter of the proteins encoded by the human genome but for up to two thirds of known druggable targets, highlighting their critical pharmaceutical importance. Hence, defining the function of MPs at a molecular level and obtaining high-resolution structural information is a crucial issue; unfortunately, it has remained a difficult task to prepare and crystallize sufficient quantities of MPs for structural analysis. Heterologous expression systems have been significantly improved in the recent years, and the challenge is now to translate these improvements to crystal structures. This chapter begins with an overview of the potential of the yeast Saccharomyces cerevisiae as a host for overexpression and purification of MPs for structural purposes; a variety of already-solved structures of MPs overexpressed in S. cerevisiae are presented and the methodologies used to obtain these structures are discussed. It also examines the case of the rabbit Ca2+-ATPase Serca1a, which was expressed in S. cerevisiae in our laboratory, and describes the critical steps that led to the determination of the three-dimensional structure of this MP. Finally, expression, solubilization, stability in detergent, and functional characterization of a complex of two MPs, namely the lipid transporter (“flippase”) complex Drs2p/Cdc50p, is presented.

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Acknowledgments

We wish to thank Raphaëlle Barry for her initial help in the “flippase” project, and specifically for construction of the co-expression plasmid, and Stéphanie David-Bosne for stimulating discussions.

This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) and by grants from the Agence Nationale pour la Recherche and the Ile de France region (Domaine d’Intérêt Majeur Maladies Infectieuses, DIM MALINF).

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  1. Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, 91405, Gif-sur-Yvette, France

    Cédric Montigny, Hassina Azouaoui, Aurore Jacquot, Marc le Maire, Christine Jaxel, Philippe Champeil & Guillaume Lenoir

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  1. Cédric Montigny
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Correspondence to Guillaume Lenoir .

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  1. UMR-CNRS 7275, Institute of Molecular and Cellular Pharmacology, Valbonne, France

    Isabelle Mus-Veteau

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Montigny, C. et al. (2014). Overexpression of Membrane Proteins in Saccharomyces cerevisiae for Structural and Functional Studies: A Focus on the Rabbit Ca2+-ATPase Serca1a and on the Yeast Lipid “Flippase” Complex Drs2p/Cdc50p. In: Mus-Veteau, I. (eds) Membrane Proteins Production for Structural Analysis. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-0662-8_6

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