# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 4 Discussion
Indeed, our FRAP data for G\(\gamma\) subunits correlated well with translocation rates in a G\(\gamma\)-dependent manner only for heterotrimers composed of G\(\alpha\)i\({}_{3}\). The mobility of G\(\alpha\)i\({}_{3}\)\(\beta_{1}\gamma_{9}\) is much higher than that of G\(\alpha\)i\({}_{3}\)\(\beta_{1}\gamma_{2}\) and its population in the membrane is also lower. On the contrary, G\(\beta_{1}\gamma_{2}\) associated with G\(\alpha\)s diffuses faster than G\(\alpha\)s\(\beta_{1}\gamma_{9}\). In fact, both G\(\alpha\)s\(\beta\gamma\) heterotrimersdiffused significantly slower when compared with their G\(\alpha\)i\({}_{3}\) associates. At the same time, if the concentrations of both the G\(\alpha\)s heterotrimers at the plasma membrane are comparable, the reduced mobility of G\(\alpha\)s\({}_{1}\gamma\)\({}_{9}\) cannot be considered to be the result of a higher proportion of uncomplexed G\(\alpha\)s. The diffusion data thus indicated that the membrane localization of G-proteins was dependent not only on G\(\beta\gamma\) but also on the G\(\alpha\) subtype since distinct heterotrimeric combinations showed different mobility characteristics. The G\(\alpha\) subunits are palmitoylated and mostly myristoylated, depending on the specific G\(\alpha\)-subclass. The dual, N-palmitoylation and S-palmitoylation, of G\(\alpha\)s is similar to the N-myristoylation and S-palmitoylation motif of the G\(\alpha\)i class, but they differ in the number of positive charge residues at the N-terminus [6; 49]. The membrane binding area of G\(\alpha\)s or G\(\alpha\)i\({}_{3}\) is limited to two sites on the surface of the protein and the membrane [50]. However, most of the membrane binding area of G\(\alpha\) is formed by the N-terminus with covalently attached lipids. Consequently, since the IEK mutation reduces palmitoylation, apart from disrupting G\(\beta\)\({}_{1}\) coupling, it also affects proper membrane localization of G\(\alpha\)s. Since this mutation reduces palmitoylation, it affects the specific binding of G\(\alpha\)s to the plasma membrane [23]. Indeed, our diffusion data suggest that the N-termini residues of G\(\alpha\)s function as an essential signal to ensure the correct localization of the subunit at the plasma membrane. Unlike for the heterotrimers formed by the wild-type G\(\alpha\)s and G\(\alpha\)i\({}_{3}\), the diffusion coefficient of distinct G\(\alpha\)s\({}_{\text{IEK}}\beta\)\({}_{1}\gamma\)\({}_{2}\) and G\(\alpha\)s\({}_{\text{IEK}}\beta\)\({}_{1}\gamma\)\({}_{9}\) heterotrimers is equal, as is the intracellular cAMP concentration, indicating that the mutation eliminates the specificity of the G\(\alpha\)s\({}_{\text{IEK}}\) heterotrimers. However, it is difficult to clearly indicate whether the substitution of charged residues in the G\(\beta\gamma\) interacting surface or the reduced palmitoylation impairs the specificity of heterotrim membrane targeting. Nevertheless, the presented results support the notion of the presence of membrane attachment signals in the N-termini of G\(\alpha\) subunits. As we previously reported, preferences in localization within the membrane of the stimulatory and inhibitory G\(\alpha\) subunits are different [25]. G\(\alpha\)s prefers solid-like domains (insensitive to cholesterol and structure or composition of lipid rafts), while G\(\alpha\)i\({}_{3}\) prefers the more fluid regions of the membrane and also detergent-resistant lipid rafts. The membrane mobility of G\(\alpha\)s is relatively slow, while the G\(\alpha\)i\({}_{3}\) diffusion is much faster. When G\(\beta\gamma\) dimer binds to the G\(\alpha\), despite the increase in the molecular weight of the complex, it accelerates the lateral diffusion of G\(\alpha\) in all tested heterotrimers. Thus, this finding again strengthens the hypothesis that the G\(\beta\gamma\) dimer not only affects the diffusion of G\(\alpha\) but also relocates complexed G\(\alpha\) within the plasma membrane.