LAUSR

Approximately 30% of the genes in the human genome code for membrane proteins, and yet we know relatively little about these complex molecules. Therefore, the biochemical and structural characterization of this challenging class of proteins represents an important frontier in both fundamental research and advances in drug discovery. However, due to their unique physical properties and requirement for association with cellular membranes, expression in heterologous systems is often daunting. In this chapter we describe how to engineer the yeast Pichia pastoris to obtain humanized sterol compositions. By implementing some simple genetic engineering approaches, P. pastoris can be reprogrammed to mainly produce cholesterol instead of ergosterol. We show how to apply mass spectrometry to confirm the production of cholesterol instead of ergosterol and how we have further analyzed the strain by electron microscopy. Finally, we delineate how to apply and test the cholesterol-forming P. pastoris strain for functional expression of mammalian Na,K-ATPase α3β1 isoform. Na,K-ATPases have been shown to specifically interact with cholesterol and phospholipids, and, obviously, the presence of cholesterol instead of ergosterol was the key to stabilizing correct localization and activity of this ion transporter.