Mutational analysis of α-subunit of protein farnesyltransferase: Evidence for a catalytic role

Protein farnesyltransferase from rat brain is composed of tightly associated α- and β-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively. The enzyme attaches farnesyl groups to cysteines in p21^ras and other proteins that contain cysteine residues at the fourth position from the COOH terminus. Production of stable enzyme in animal cells requires the simultaneous synthesis of both subunits, and all activity is lost when the subunits are dissociated chemically. The β-subunit functions in the Zn²⁺-dependent binding of the protein substrate. The role of the α-subunit is unknown. In the current studies we used in vitro mutagenesis and transfection of cloned cDNAs to define the parts of the α-subunit that are necessary to stabilize the β-subunit and to support farnesyl transfer. Deletion of 51 amino acids at the NH₂ terminus of the α-subunit allowed normal stabilization of the β-subunit and production of normal enzyme activity, but deletion of 106 amino acids abolished both of these properties. A proline-rich region at residues 12-34 of the α-subunit is not required for activity, but its presence explains the anomalously slow migration of the polypeptide on SDS-polyacrylamide gels. Deletion of only 5 amino acids at the COOH terminus of the α-subunit reduced activity appreciably. Substitution of asparagine for a conserved lysine at position 164 produced an α-subunit that stabilized the β-subunit normally and permitted normal binding of the two substrates, farnesyl pyrophosphate and p21^H-ras. Nevertheless, the rate of transfer of the bound farnesyl group to p21^H-ras was markedly reduced. The latter finding suggests that the α-subunit plays a direct role in the catalytic reaction in addition to its role in the stabilization of the β-subunit.