title: Function of positive charges following signal-anchor sequences during translocation of the N-terminal domain.
abstract: In topogenesis of membrane proteins on the endoplasmic reticulum, the orientation of the hydrophobic transmembrane (TM) segment is influenced by the charge of the flanking amino acid residues. We assessed the function of the positive charges downstream of the hydrophobic segment using synaptotagmin II. The positive charges were systematically replaced with non-charged residues. Although the original TM segment translocated the N terminus, the topology was inverted, depending on the mutations. Orientation was affected in mutants in which 6 Lys were shifted downstream, even when the 6 Lys were 25 residues from the hydrophobic segment. The Lys was functionally replaced by Arg, but not by Asp or Glu. The timing of action during polypeptide elongation indicated that the Lys functions at the ribosome exit sites. We suggest that the commitment of the TM segment to a particular orientation is influenced by far downstream parts of the polypeptide chain and that the positive charges are decoded after exiting the ribosome.
authors.affiliation: Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency, Hyogo.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Kida
authors.forename: Fumiko
authors.initials: F
authors.lastname: Morimoto
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Mihara
authors.forename: Masao
authors.initials: M
authors.lastname: Sakaguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D016000:Cluster Analysis; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D018698:Glutamic Acid / Q000737:chemistry; D006031:Glycosylation; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D050861:Synaptotagmin II / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D050861:Synaptotagmin II; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D003513:Cycloheximide; D008239:Lysine