Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae.
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.
Flegelova
Hana
H
Department of Membrane Transport, Institute of Physiology AS CR, Videnska 1083, 142 20, Prague 4, Czech Republic.
Haguenauer-Tsapis
Rosine
R
Sychrova
Hana
H
D000818:Animals; D003001:Cloning, Molecular; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000235:genetics; D010641:Phenotype; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000235:genetics
D050506:Mutant Chimeric Proteins; D011994:Recombinant Proteins; C081517:SLC9A2 protein, human; C000613385:SLC9A3 protein, human; C452850:Slc9a1 protein, rat; C513110:Slc9a2 protein, rat; C000613387:Slc9a3 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D005990:Glycerol