Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: up-regulation by a phosphorylated cytosolic protein (ReP1-NCXSQ) is identical to that of native exchanger in situ

Abstract

This work shows, for the first time, a properly metabolically regulated squid nerve Na(+)/Ca(2+) exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST-6HB-NCXSQ1-eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca(2+) with the expected characteristics of the reverse Na(+)/Ca(2+) exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca(2+)(i) regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1-NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1-NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1-NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST-6HB-NCXSQ1-eGFP protein, Na(+)/Ca(2+) exchange was stimulated by phosphorylated ReP1-NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.