Heterogeneous processing of a G protein gamma subunit at a site critical for protein and membrane interactions.

The G protein gamma5 subunit is selectively associated with specific G protein alpha subunits [Wilcox, M. D., et al. (1995) J. Biol. Chem. 270, 4189] and is localized preferentially in focal adhesion plaques [Hansen, C. A., et al. (1996) J. Cell Biol. 126, 811]. What determines the differential association of G proteins and their subunits with specific cellular structures or compartments is not clear, but one factor could be variation in the pattern of processing of the proteins. To study gamma5 subunit diversity and modifications, G protein subunits were fractionated on an HPLC phenyl column and analyzed with a gamma5-specific antiserum. The gamma5 eluted from the column as two peaks of immunoreactivity. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray ionization tandem mass spectrometry revealed that the first immunoreactive peak corresponded to the predicted gamma5 isoform (N-terminally acetylated after removal of methionine, C-terminally geranylgeranylated and carboxymethylated with removal of the last three amino acids), while the second peak of immunoreactivity contained a gamma5 isoform isoprenylated at the C-terminus but retaining its three terminal amino acids. This alternatively processed protein is the predominant gamma5 subunit isoform associated with Go and Gi proteins purified from bovine brain. These results describe a new C-terminal processing pattern for G protein gamma subunits and establish the principle that G protein gamma subunits can be heterogeneously modified at their C-termini. This is a site on the gamma subunit critical for membrane and protein-protein interactions of G proteins. These results open the possibility that one determinant of the localization of G proteins in cells could be the pattern of processing of their gamma subunit constituents.