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Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

By Hana Flegelova, Rosine Haguenauer-Tsapis and Hana Sychrova

Abstract

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane

Topics: [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology
Publisher: HAL CCSD
Year: 2006
DOI identifier: 10.1016/j.bbagen.2006.01.014
OAI identifier: oai:HAL:hal-00067955v1
Provided by: Hal-Diderot
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