NCX1 expressed in S. cereviseae show similar interaction with PtdIns-4,5-P2 as native NCX1

RAIMUNDA, DANIEL; POSADA, VELIA; BEAUGÉ, LUIS; BERBERÍAN, GRACIELA

Uruguay

Congreso; 6th. International Conference of Biological Physics - 5th. Southern Cone Biophysics Congress – Biophysical Society of Argentina XXXVI Annual Meeting; 2007

Previous results from our laboratory have shown that in bovine heart vesicles the forward mode of the Na+/Ca2+ exchanger (NCX1) is stimulated by physiological concentrations of Mg-ATP at limiting [Ca2+]i. This process is mediated by an increment of phosphatidylinositol-4,5-biphosphate (PtdIns-4,5-P2) bound to or in the in micro-environment surrounding the exchanger. The aim of the present work was to find structures or domains of the exchanger that could be involved in the binding of PtdIns-4,5-P2. For this purpose we expressed the NCX1 with a12 histidine tag in the budding yeast S. Cerevisiae. The exchanger, purified with a Ni2+-NTA agarose resin, was functional. Two techniques were used to asses the NCX1-PtdIns-4,5-P2 interaction: (i) Western blot with co-inmunolabeling with anti-PtdIns-4,5-P2 and anti-NCX1 antibodies, and (ii) Dot blot binding in PVDF strips with several membrane lipids including poly-phosphoinositides from both synthetic and natural sources. As occurred in bovine heart vesicles, the level of PtdIns-4,5-P2 “bounded” to NCX1-12H was dounbled when yeast microsomes were exposed to 1mM Mg-ATP at 1μM free Ca2+; this correlates well with the increment in the Na+-Ca2+ exchange activity observed in bovine heart vesicles. To further characterize the lipid-protein interaction 80% pure protein (detected by silver stain) was incubated with phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P) and PtdIns-4,5-P2 dotted strips and then analyzed by revealing them with anti-NCX1 antibody. Surprisingly, the exchanger protein showed specific binding to PtdIns-4,5-P2 from natural sources (bovine brain) but an unspecific binding to several phosphatidylinositides and others negatively charged lipids from synthetic sources (commercial strips). Despite this unexpected result, we have demonstrated that, as it happens with the NCX1 present in sarcolemmal vesicles, the functionally expressed NCX1-12H in yeast plasma membranes binds PtdIns-4,5-P2. We expect that this approach will allow us to further characterize the binding domain/s of PtdIns-4,5-P2 in the NCX1 protein.