Characterization and localization of prenylcysteine carboxymethyltransferase in the SH-SY5Y neuroblastoma cell line: The methyltransferase is in the endoplasmic reticulum

Abstract

Membranes of SH-SY5Y neuroblastoma cells contain a prenylcysteine carboxymethyltransferase that methylates the pseudosubstrates AFC (N-acetylfarnesylcysteine, Km = 127 μM) and AGGC (N-acetylgeranylgeranylcysteine, Km = 12μM) as well as some endogenous membrane proteins. Two techniques were used to demonstrate the methylation of endogenous proteins. While the vapor phase equilibrium-assay revealed only the alkaline-labile methylated membrane proteins that were mainly localized in the molecular mass (MM) range of 21-25 kDa, all methylated proteins were detected using the P-screen method. However, the peak consisting of methylated proteins of the MM of 21-25 kDa exceeded by far the peaks of other proteins using the latter method. The effect of inhibitors of endogenous methylation was tested and both methods gave similar results, thus validating the use of the P-screen method. With the latter, a Km value of 0.8 μM was determined for AdoMet (S-adenosyl-L-methylmethionine; methylation of the 21-25 kDa proteins). Antibodies against human protein-S-prenylcysteine-O-methyltransferase (pcCMT) were raised in chicken and used to identify endogenous protein-S-prenylcysteine-O-methyltransferase (pcCMT) in SH-SY5Y cells by immunoblotting. With these anti-pcCMT antibodies a specific 32 kDa protein could be detected in SH-SY5Y membranes. Two fractionation techniques were applied to localize the methyltransferase in SH-SY5Y cells. Applying differential pelleting and gradient centrifugation, mainly the endoplasmic reticulum proved to be a likely candidate for the subcellular localization of pcCMT in SH-SY5Y cells. Additional indirect immunofluorescence confirmed that pcCMT was expressed in the endoplasmic reticulum and could neither be clearly demonstrated in the plasma membranes nor in large dense vesicle (LDV) membranes.