In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.
The gamma subunits of the heterotrimeric G proteins (G gamma) are subject to carboxyl-terminal processing. This processing involves prenylation of a cysteine residue initially 4 amino acids from the carboxyl terminus, endoproteolytic truncation of the 3 terminal amino acids, and methylation of the now carboxyl-terminal prenylcysteine residue. The significance of each of these modifications in the ultimate properties of G proteins is not yet clear. We have developed in vitro systems for the production of the three processing intermediates (unmodified, prenylated, and truncated-prenylated) for two G gamma subunits, one which is subject to farnesylation (G gamma 1) and one which is geranylgeranylated (G gamma 2). Assessment of the functional consequences of the processing of G gamma was found to require reconstitution of the polypeptides with a G protein beta subunit (G beta). The ability of recombinant G beta, produced in Sf9 cells, to assemble into stable beta gamma complexes (G beta gamma) with each of the G gamma processing intermediates was assessed. Both prenylated and unprenylated G gamma subunits formed stable complexes with G beta, but surprisingly, neither of the truncated-prenylated G gamma subunits were competent for this assembly. The G beta gamma complexes which were formed were examined for their ability to interact with a G protein alpha subunit (G alpha). Only those G beta gamma complexes containing a prenylated G gamma subunit were functional in this assay. These data indicate that: 1) prenylation of G gamma is not required for G beta gamma assembly; 2) assembly of the G beta gamma complex occurs prior to the proteolytic processing of G gamma; and 3) G beta gamma complexes require prenylated G gamma for interaction with G alpha.
Higgins
J B
JB
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
Casey
P J
PJ
D002268:Carboxypeptidases / Q000494:pharmacology; D004122:Dimethylallyltranstransferase / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D014357:Trypsin / Q000494:pharmacology
D046911:Macromolecular Substances; D011498:Protein Precursors; D011994:Recombinant Proteins; D004122:Dimethylallyltranstransferase; D002268:Carboxypeptidases; D014357:Trypsin; D019204:GTP-Binding Proteins