Abstract
Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.
Highlights
W e report the subcellular localization and characterization of a neutrophil prenylcysteianecarboxyl methyltraasferase.The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme
Neutrophil specific granules contained one-sixth as much carboxyl methyltransferase activity as that found in the light membrane fraction and contained proportional levels of biotinylation and alkaline phosphatase activity, suggestingcontamination of the specific granule fraction with plasma membranes
Methylation of ras-related proteins increases their affinity for membranes [5]; whether this effect is due to increased hydrophobicity alone or to specific targeting by the prenylcysteine methylester remains unresolved
Summary
C A M ) where C is cysteine, A is usually an aliphatic amino acid, and X is another amino acid [1].Several cytosolic prenyltransferases recognize these sequences and catalyze the into lipid bilayers or binds to specific membrane targets. Aplasma membrane localization of the prenylcysteine-directed carboxyl methyltransferase. Samples of subcellular fractions (200 pg) were blotted onto nitrocellulose, which was blocked for 1 h at room temperature in TGGbuffer (PBS with 0.5% Tween 20, 1 M glucose, 10% glycerol) containing 3% bovine related proteins followed neutrophil activation and that spe- Serum albumin and 2% nonfat dry milk. The nitrocellulose was cific inhibitorsof prenylcysteine-directedcarboxyl methyl- washed twice in PBS, 0.5% Tween 20 and incubated for 2 h in transferase inhibited neutrophil function [23], we haverecently suggestedaroleforcarboxyl methylation of GTPbinding proteins in signal transduction. The enzyme is present at theplasmalemma, requires anionic membrane fraction, streptavidin- or cellobiose- (control) conjugatedphospholipid forreconstitutionafter detergent solubilization, agarose beads were washed with TE buffer. Membrane of neutrophils was labeled by biotinylating the primary amines on surface molecules according to themethod of Rosen et al
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