SUBCELLULAR-DISTRIBUTION AND GUANINE-NUCLEOTIDE DEPENDENCY OF COOH-TERMINAL METHYLATION IN KIDNEY CORTEX

The subcellular distribution of COOH-terminal carboxyl methyltransfera se and methylated substrates was studied in purified brush-border and basolateral plasma membranes, as well as in crude intracellular membra nes and the cytosolic fraction isolated from rat kidney cortex. The th ree membrane fractions showed intrinsic carboxyl methylation of 21- to 23-kDa proteins, whereas 18- and 41-kDa methylated proteins were obse rved in the cytosol. In contrast, methylation activities toward N-acet yl-S-trans,trans-farnesyl-L-cysteine (AFC), a synthetic farnesylated s ubstrate, were found to be strictly associated with membranes, with no detectable level of activity in the cytosol. Methylation of all membr ane-associated substrates was inhibited by AFC but remained unaffected by TS-isoD-YSKY, a synthetic isopeptide recognized by L-isoaspartyl m ethyltransferase, suggesting that the membrane-associated substrates w ere methylated on a COOH-terminal isoprenylated cysteine residue. The membrane-associated methylated proteins were tightly bound to the memb ranes as reflected by their extraction with olamidopropyl)-dimethylamm onio]-1-propanesulfonate but not with 1 M NaCl or 2 M urea. The nonhyd rolyzable analogues of GTP and GDP, guanosine 5'-O-(3-thiotriphosphate ) (GTP-gammaS) and guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), mark edly increased the methylation of the 21- to 23-kDa substrates, wherea s ATPgammaS and ADPbetaS were without effect. This effect of guanine n ucleotides was restricted to endogenous 21- to 23-kDa substrates with no stimulation of methylation of the exogenous substrate, AFC. Our res ults show a wide distribution of both COOH-terminal protein carboxyl m ethyltransferase activities and associated methylated substrates in th e kidney cortex. Moreover, the variation in the degree of methylation of the membrane-associated 21- to 23-kDa proteins by guanine nucleotid es may suggest the existence of cellular mechanisms involved in the re gulation of the methylation activities.