The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine.
Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyl]methionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the alpha subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory gamma subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the alpha cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the alpha-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.
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Jules Stein Eye Institute, University of California School of Medicine, Los Angeles.
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Clarke
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Fung
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D008745:Methylation; D010446:Peptide Fragments / Q000302:isolation & purification; D010786:Photoreceptor Cells / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; D003545:Cysteine