A NOVEL ENDOPROTEASE RESPONSIBLE FOR THE SPECIFIC CLEAVAGE OF TRANSDUCIN GAMMA-SUBUNIT

Isoprenylated/methylated heterotrimeric G proteins play important role s in a large number of signal transduction processes. While the enzymo logy of isoprenylated/methylated protein biosynthesis is well understo od, nothing is known about how these proteins are degraded. In this ar ticle, a novel endoproteolytic activity has been identified from bovin e retina and is shown specifically to remove the glycylfarnesylcystein e moiety from the carboxyl terminus of T-gamma. When tested in a GTP b inding assay, freshly prepared proteolyzed T-beta gamma, was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-ga mma-S) to T-alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and t he pH profile corresponds to an enzyme having pK(a)'s of 4.4 +/- 0.1 a nd 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhib itor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity l abeling studies using biotinylated affinity labeling probes have ident ified a 35 kDa protein as a candidate for the endoprotease.