A hydrophobic region locating at the center of fibroblast growth factor-9 is crucial for its secretion.
Fibroblast growth factor (FGF)-9 is a glycosylated neurotrophic polypeptide highly expressed in brain. The mechanism for its secretion from expressing cells is unclear, because its primary structure lacks a cleavable signal sequence. We, therefore, investigated the mechanism and structural requirements for secretion of FGF-9. As with other secreted proteins, in vitro translation of FGF-9 was inhibited by signal recognition particle, which binds to the signal sequence. When translated in vitro, full-length FGF-9 was translocated into microsomes, glycosylated, and protected from trypsin digestion. By using various FGF-9 deletion mutants, we found that two hydrophobic domains, located at the N terminus and at the center of the FGF-9 primary structure, were crucial for translocation. Examination of various point mutants revealed that local hydrophobicity of the central hydrophobic domain, but not the N terminus, was crucial for translocation. Analogous results were obtained with respect to FGF-9 secretion from transfectant cells. Upon deletion of the complete sequence preceding it, the previously uncleavable hydrophobic domain appeared to serve as a cleavable signal sequence. Our results suggest that nascent FGF-9 polypeptides translocate into endoplasmic reticulum without peptide cleavage via a co-translational pathway in which both the N terminus and the central hydrophobic domain are important; thereafter, FGF-9 is glycosylated and secreted.
Miyakawa
K
K
Biosignaling Department, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305-8566, Japan.
Hatsuzawa
K
K
Kurokawa
T
T
Asada
M
M
Kuroiwa
T
T
Imamura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017931:DNA Primers; D051525:Fibroblast Growth Factor 9; D005346:Fibroblast Growth Factors; D006031:Glycosylation; D006133:Growth Substances / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes; D018271:Signal Recognition Particle / Q000378:metabolism; D014162:Transfection; D014908:Triticum
D017931:DNA Primers; C496461:Fgf9 protein, rat; D051525:Fibroblast Growth Factor 9; D006133:Growth Substances; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle; D005346:Fibroblast Growth Factors