Gαi3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins

G alpha i3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both G alpha i3 and Ca2+ and is found both in the Golgi lumen and in the cytoplasm. To investigate whether G alpha i3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between G alpha i3 and calnuc in COS-7 cells expressing G alpha i3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When G alpha i3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when G alpha i3 is coexpressed with cytosolic calnuc(Delta N2-25)-CFP lacking its signal sequence. No FRET signal is detected when G alpha i3(Delta C12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when G alpha i3-YFP and calnuc(Delta EF-1,2)-CFP, which is unable to bind G alpha i3, are coexpressed. G alpha i3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to G alpha i3 on Golgi membranes in living cells and that G alpha i3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca2+ sensor protein capable of binding to and potentially regulating interactions of G alpha i3 on Golgi membranes.