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

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 yeastSaccharomyces cerevisiae as a host for overexpression and purification of MPs for structural purposes; a variety of already-solved structures of MPs overexpressed inS. 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 inS. 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 presente.