title: Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane.
abstract: Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
authors.forename: T
authors.initials: T
authors.lastname: Kuroiwa
authors.forename: M
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: K
authors.initials: K
authors.lastname: Mihara
authors.forename: T
authors.initials: T
authors.lastname: Omura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D012995:Solubility
chemical_list: D007376:Interleukin-2; D008565:Membrane Proteins; D004247:DNA