The sarco/endoplasmic reticulum calcium ATPase (SERCA) has been proposed to form functional dimers in vitro. In order to investigate whether SERCA forms homo-dimers in live cells, we fused canine SERCA2a to cerulean (Cer) or yellow fluorescent protein (YFP), and quantified SERCA-SERCA interactions by fluorescence resonance energy transfer (FRET). SERCA-SERCA FRET efficiency was dependent on the labeling position of the fluorescent protein tags, with the highest FRET efficiency achieved when the respective fluorescent proteins were fused to SERCA N-termini. FRET was reduced by competition with unlabeled SERCA, suggesting that the observed FRET was due to specific protein-protein interactions. Progressive photobleaching of YFP showed that Cer intensity increased linearly with decreasing YFP intensity, suggesting that the stoichiometry of the SERCA complex is a dimer. In contrast, a control experiment with phospholamban (PLB) oligomer showed a non-linear YFP/Cer relationship, consistent with its well-known pentameric stoichiometry. We also investigated whether SERCA dimers could interact with PLB, the regulatory binding partner of SERCA. Interestingly, while average maximal FRET was 28% between SERCA and PLB, fluorescence lifetime measurements revealed two different lifetimes, consistent with two different populations of FRET donors. One population showed very low FRET, while the other population exhibited high FRET- approximately double the measured average maximal FRET efficiency. The data are consistent with a single PLB bound to each SERCA homo-dimer; in this regulatory complex one SERCA protomer is in close proximity to PLB (50 Å), while the other is too far away to participate in FRET with PLB.
Quantitative Measurement of Fluorescence Resonance Energy Transfer (FRET) Efficiency
