The N‐terminus of B96Bom, a Bombyx mori G‐protein‐coupled receptor, is N‐myristoylated and translocated across the membrane

In eukaryotic cellular proteins, protein N‐myristoylation has been recognized as a protein modification that occurs mainly on cytoplasmic or nucleoplasmic proteins. In this study, to search for a eukaryotic N‐myristoylated transmembrane protein, the susceptibility of the N‐terminus of several G‐protein‐coupled receptors (GPCRs) to protein N‐myristoylation was evaluated by in vitro and in vivo metabolic labeling. It was found that the N‐terminal 10 residues of B96Bom, a Bombyx mori GPCR, efficiently directed the protein N‐myristoylation. Analysis of a tumor necrosis factor (TNF) fusion protein with the N‐terminal 90 residues of B96Bom at its N‐terminus revealed that (a) transmembrane domain 1 of B96Bom functioned as a type I signal anchor sequence, (b) the N‐myristoylated N‐terminal domain (58 residues) was translocated across the membrane, and (c) two N‐glycosylation motifs located in this domain were efficiently N‐glycosylated. In addition, when Ala4 in the N‐myristoylation motif of B96Bom90‐TNF, Met‐Gly‐Gln‐Ala‐Ala‐Thr(1–6), was replaced with Asn to generate a new N‐glycosylation motif, Asn‐Ala‐Thr(4–6), efficient N‐glycosylation was observed on this newly introduced N‐glycosylation site in the expressed protein. These results indicate that the N‐myristoylated N‐terminus of B96Bom is translocated across the membrane and exposed to the extracellular surface. To our knowledge, this is the first report showing that a eukaryotic transmembrane protein can be N‐myristoylated and that the N‐myristoylated N‐terminus of the protein can be translocated across the membrane.