Abstract
The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure–function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [3H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.
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Acknowledgments
We thank Laura Popolo for sending us the P. pastoris S-α3β1 strain and the human Na,K-ATPase α3β1 isoform expression plasmid, Steven Karlish for the generous gift of anti-KETYY and anti-GERK antibodies, Guenther Daum for providing his laboratory for radioactive experiments and for the Pma1p antibody, Gloria Padoani for kind assistance via e-mail, and Helmut Schwab for valuable advice. H.P. acknowledges support by a NAWI Graz GASS project.
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Hirz, M., Richter, G., Leitner, E. et al. A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform. Appl Microbiol Biotechnol 97, 9465–9478 (2013). https://doi.org/10.1007/s00253-013-5156-7
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DOI: https://doi.org/10.1007/s00253-013-5156-7