title: Systematic analysis of stop-transfer sequence for microsomal membrane.
abstract: Co-translational protein translocation across the endoplasmic reticulum membrane is interrupted by particular amino acid sequences which are called stop-transfer sequences. Since the stop-transfer process should reflect the character of the protein translocation machinery, systematic examination on the structural requirements for stop-transfer sequences should give information about the translocation process. By the manipulation of the cDNA of interleukin 2, a typical secretory protein, the middle portion of the molecule was replaced with systematically constructed hydrophobic stretches, and two positively or negatively charged amino acid residues were introduced just behind the hydrophobic stretches. The modified proteins were synthesized with an in vitro transcription-translation system in the presence of dog pancreas rough microsomes, and their topologies in the membrane were examined with proteinase K digestion. The efficiency of stop-translocation depended on the hydrophobicity and the length of the inserted stretch. The segments followed by positively charged residues interrupted the translocation more efficiently than those with negatively charged residues. We observed that more than 19 alanine residues were required for efficient stop-translocation, whereas only 9 leucine residues were sufficient. We suggest that the positively charged residues following the hydrophobic stretches promote stop-translocation of the peptides through the channel.
authors.affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
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; D001692:Biological Transport; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D007376:Interleukin-2; D008562:Membrane Glycoproteins; D012333:RNA, Messenger