标题：Gαi3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2001
卷号：98
期号：26
页码：14961-14966
DOI：10.1073/pnas.261572098
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Gi3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Gi3 and Ca2+ and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Gi3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Gi3 and calnuc in COS-7 cells expressing Gi3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Gi3-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 Gi3 is coexpressed with cytosolic calnuc({Delta}N2-25)-CFP lacking its signal sequence. No FRET signal is detected when Gi3({Delta}C12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Gi3-YFP and calnuc({Delta}EF-1,2)-CFP, which is unable to bind Gi3, are coexpressed. Gi3(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 Gi3 on Golgi membranes in living cells and that Gi3 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 Gi3 on Golgi membranes.
