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.
Utsumi
Toshihiko
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. utsumi@yamaguchi-u.ac.jp
Ohta
Hiroto
H
Kayano
Yoshiyuki
Y
Sakurai
Nagisa
N
Ozoe
Yoshihisa
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism
D019476:Insect Proteins; D043562:Receptors, G-Protein-Coupled; D019814:Myristic Acid