IN-VITRO ASSAY AND CHARACTERIZATION OF THE FARNESYLATION-DEPENDENT PRELAMIN-A ENDOPROTEASE

The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa f arnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of ov erexpressed wild-type and mutant lamin A proteins in cultured cells ha ve indicated that the precursor possesses the typical carboxyl-termina l S-farnesylated, cysteine methyl ester and that farnesylation is requ ired for endoproteolysis to occur. In this report, we describe the syn thesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. T his peptide acts as a substrate for the prelamin A endoprotease in vit ro, with cleavage of the synthetic peptide at the expected site betwee n Tyr(657) and Leu(658). Endoproteolytic cleavage requires the S-preny lated cysteine methyl ester and, in agreement with transfection studie s, is more active with the farnesylated than geranylgeranylated cystei nyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a nonc ompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.