# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 3 Results
### Analysis of G-Protein Interactions in Live Cells Using FLIM-FRET Microscopy  
FLIM-FRET was employed to assess whether the G\(\gamma\) subunits played a role in plasma membrane localization and formation of the G-protein heterotrimer. For these reasons, we performed experiments to evaluate the molecular interactions of two dimers, G\(\beta_{1}\gamma_{2}\) and G\(\beta_{1}\gamma_{9}\), with G\(\alpha\)s and G\(\alpha\)i\({}_{3}\). As we have reported, these two different G\(\alpha\) subclasses show different lipid preferences and, consequently, are localized in distinct types of membrane domains [25]. Thus, we examined here if heterotrim formation was also controlled in a G\(\alpha\)-subclass dependent manner. We monitored FRET by measuring the lifetime of the donor fluorophore (Citrine) in the absence and presence of the acceptor (mCherry), as described in the Materials and Methods section. In our experimental system, Citrine (Citrine-G\(\alpha\)i\({}_{3}\beta_{1}\gamma_{2}\), Citrine-G\(\alpha\)s\({}_{1}\gamma_{2}\)) exhibited a double exponential decay with lifetimes of \(2.7\pm 0.1\) ns (\(\tau_{1}\)) and \(3.4\pm 0.15\) ns (\(\tau_{2}\)), implying the existence of two donor species. The amplitude of each of these lifetimes was approximately 50%. The presence of multi-exponential fluorescence decays in various intrinsically FP such as Citrine, cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), monomeric green fluorescent protein (mGFP), and Discosoma red fluorescent protein (DsRed) has been reported in several articles [34; 35; 36]. In the FRET system (the cells expressing Citrine-G\(\alpha\) were additionally transfected with mCherry-G\(\beta_{1}\) and the appropriate G\(\gamma\)), the donor emission curves were also fitted to a double exponential decay model. However, a shortening of the fluorescence lifetime due to FRET was observed only for the short component \(\tau_{1}\), while \(\tau_{2}\) remained almost unchanged (compared to the donor alone). This indicates that only one donor species (characterized by lifetime \(\tau_{1}\)) underwent FRET (FRETing donor fluorophore state) and that the other species with the longer lifetime (\(\tau_{2}\)) was not engaged in FRET (non-FRETing donor fluorophore state). Therefore, only the FRETing component was used to calculate FRET efficiency (Figure 2C). Lifetime shortening due to FRET was also observed in the FLIM images as a uniform change in color toward the blue hues across all pixels (Figure 2A). As shown in Figure 2B, different combinations of G\(\alpha\) subunits and G\(\beta_{1}\gamma\) dimers gave varying levels of donor lifetime changes (box chart), and thus unequal FRET efficiencies. The highest FRET efficiency of 50% was obtained in cells expressing G\(\alpha\)i\({}_{3}\beta_{1}\gamma_{2}\) and the efficiency was only 5% lower for the G\(\alpha\)i\({}_{3}\beta_{1}\gamma_{9}\) heterotrimer. In contrast to Citrine-G\(\alpha\)i\({}_{3}\), Citrine-G\(\alpha\)s exhibited a lower FRET signal when paired with either mCherry-G\(\beta_{1}\gamma_{2}\) or mCherry-G\(\beta_{1}\gamma_{9}\). The lifetime of Citrine-G\(\alpha\)s in cells co-expressing the mCherry-G\(\beta_{1}\gamma_{2}\) dimer was found to be \(2.12\pm 0.21\) ns, amounting to 19.7% energy transfer efficiency, and a similar value was obtained for the complex with the mCherry-G\(\beta_{1}\gamma_{9}\) dimer, giving a FRET signal of 19.5%. An important question is whether the differences in FRET efficiency we observed are a measure of the efficiency of association between the interacting proteins or a consequence of structural differences between the heterotrimers. In the case of two proteins labeled with donor and acceptor molecules, FRET is highly dependent not only on the distance between the donor and acceptor but also on the stoichiometry of macromolecular interactions as well as on the donor fraction taking part in complex formation with acceptors.