# G\(\gamma\) and G\(\alpha\) Identity Dictate a G-Protein Heterotrim Plasma Membrane Targeting
## 4 Discussion
Notably, it is evident in the case of G\(\alpha\)s heterotrimers because the apparent diffusion coefficient is more than 1.5 times greater than for the uncomplexed subunit. As demonstrated previously, the G\(\beta\gamma\) dimer is responsible for the rapid relocation of G\(\alpha\) from the lamellar membrane region where it resides as a monomer [25; 51]. The G\(\beta\gamma\) complex remains associated with the non-lamellar regions which may explain the acceleration of the diffusion rate of heterotrimers compared to monomeric G\(\alpha\)[51]. Interestingly, as mentioned above, the lateral mobility of the G-proteins composed of the G\(\beta\)\({}_{1}\gamma\)\({}_{2}\) dimer differs significantly from those containing G\(\beta\)\({}_{1}\gamma\)\({}_{9}\) dimer; the diffusion rates of distinct heterotrimers are characterized by different values of diffusion coefficients. Hence, while the G\(\beta\gamma\) dimer defines the affinity of the complete heterotrimer for the lipid phase, the differences in the preynl moieties are not sufficient to explain the differential diffusion of the full heterotrimers. Our data correspond with the FRET-clustering analysis of fluorescently-tagged heterotrimeric G-protein-derived membrane anchors [52]. The authors have shown that the N-terminal sequences of G\(\alpha\)i/o and G\(\alpha\)q and their heterotrimers (the N-terminal part of G\(\alpha\)i\({}_{2}\) or G\(\alpha\)q merging with the C-terminal part of G\(\gamma\)\({}_{2}\)) were clustered together in different domains. Moreover, postulated membrane domains partially share their area causing overlapping domains, thus strengthening co-clustering. Upon activation the heterotrimers dissociate and the G\(\alpha\) subunits displace into a subclass-specific domains. Our FRAP data do not support the pre-activation co-clustering of the distinct heterotrimeric G-proteins. A possible explanation for this discrepancy in conclusions correlates with the use of membrane anchors of G\(\alpha\) and G\(\gamma\) subunits but not, as in the present study, with full-length G-proteins. In addition to those previously identified, both components, G\(\beta\gamma\) dimer and G\(\alpha\), determine final membrane localization of the full heterotrim. These findings imply that the dissociation of the G-protein on activation and subsequent re-association on deactivation, are also influenced by the subclass of the G\(\alpha\) subunit. Yet, the FRAP measurements could not precisely resolve membrane distribution of G-protein but strongly suggested the possibility of different localizations at the plasma membrane of particular heterotrimers (membrane domains/regions differing in lipid composition and properties). The results presented here indicate that the diffusion rates of heterotrimers composed of different G\(\beta\gamma\) and G\(\alpha\) subunits were not directly related to the membrane dissociation G\(\gamma\)-pattern that were determined by the nature of the prenyl group and by basic residues in the C termini of G\(\gamma\). The role of the G\(\alpha\) subunit in determining the membrane localization by interacting with lipids and G\(\beta\gamma\) dimers shown in the present study suggests that the G\(\gamma\) translocation rate can, consequently, be also affected by the G\(\alpha\) subunit. The mobility of the G-proteins and dissociated subunits is heterogeneous, suggesting non-random distribution within the cell membranes, which may strongly reflect the natural functions of these proteins. Many studies have emphasized the importance of the clustering of membrane proteins in a manner dependent on their functional state [53; 54]. Results obtained in the present study show that divergent heterotrimers localize to distinct membrane locations due to the combined lipid modifications on G\(\alpha\) and G\(\gamma\), together with a different number and distribution of adjacent positively charged residues (i.e., various classes of G-proteins located in distinct domains relocate upon activation and dissociation into subunits).