Human erythrocyte protein 4.2, a high copy number membrane protein, is N-myristylated.
Band 4.2 is a major protein of the erythrocyte membrane which has been immunologically detected in a variety of cell types and is apparently essential for normal erythrocyte membrane function. Since band 4.2 has unusual solubility and membrane binding properties and has an N-terminal glycine following the initiating methionine, we explored the possibility that band 4.2 is myristylated. When Sf9 cells infected with a recombinant band 4.2 Baculovirus were incubated with [3H]myristic acid, label became incorporated into recombinant band 4.2 protein and resisted extraction with hydroxylamine. Consistent with N-terminal myristylation, the incorporation of label was dependent upon protein synthesis. The fatty acid covalently bound to recombinant band 4.2 was definitively identified as myristic acid by recovering the fatty acid after hydrolysis of band 4.2 and examining its migration relative to standards in thin layer chromatography. It was determined that native erythrocyte band 4.2 is an N-myristylated protein by reverse phase high performance liquid chromatography detection of an azlactone derivative of N-myristylglycine after mild acid hydrolysis and azlactone derivatization of the purified protein. Study of myristylation of band 4.2, an abundant normal cellular protein, and its role in membrane binding may produce insights relevant to other myristylated cellular proteins.
Risinger
M A
MA
Department of Biomedical Research, St. Elizabeth's Hospital, Boston, Massachusetts 02135.
Dotimas
E M
EM
Cohen
C M
CM
D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003598:Cytoskeletal Proteins; D005455:Fluorescent Antibody Technique; D006801:Humans; D007313:Insecta; D008565:Membrane Proteins; D008715:Methionine / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D014162:Transfection
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009227:Myristic Acids; C044791:erythrocyte membrane band 4.2 protein; D019814:Myristic Acid; D008715:Methionine