title: The code for directing proteins for translocation across ER membrane: SRP cotranslationally recognizes specific features of a signal sequence.
abstract: The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP-signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N(ε)-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) amber suppressor. A homogeneous population of SRP-ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP-signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP-signal sequence affinity and targeting to the translocon.
authors: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden.
authors: IngMarie
authors: I
authors: Nilsson
authors: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden.
authors: Patricia
authors: P
authors: Lara
authors: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden.
authors: Tara
authors: T
authors: Hessa
authors: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA; Department of Chemistry, Texas A&M University, College Station, TX 77843, USA; Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors: Arthur E
authors: AE
authors: Johnson
authors: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden; Science for Life Laboratory, Stockholm University, Box 1031, 171 21 Solna, Sweden.
authors: Gunnar
authors: G
authors: von Heijne
authors: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA. Electronic address: andrey.karamyshev@utsouthwestern.edu.
authors: Andrey L
authors: AL
authors: Karamyshev
mesh_terms: D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011388:Prolactin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D012343:RNA, Transfer / Q000378:metabolism; D000069816:SEC Translocation Channels; D017384:Sequence Deletion; D018271:Signal Recognition Particle / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; C017671:preprolactin; D011388:Prolactin; D012343:RNA, Transfer
keywords: SRP; photocrosslinking; protein targeting; protein translocation; signal sequence