The Code for Directing Proteins for Translocation Across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

J Mol Biol. 2015 Mar 27;427(6 Pt A):1191-201. doi: 10.1016/j.jmb.2014.06.014. Epub 2014 Jun 28.


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.

Keywords: SRP; photocrosslinking; protein targeting; protein translocation; signal sequence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dogs
  • Endoplasmic Reticulum / metabolism*
  • Membrane Proteins / metabolism
  • Prolactin / chemistry
  • Prolactin / metabolism
  • Protein Binding / genetics
  • Protein Interaction Domains and Motifs / genetics
  • Protein Precursors / chemistry
  • Protein Precursors / metabolism
  • Protein Sorting Signals* / genetics
  • Protein Transport
  • RNA, Transfer / metabolism
  • SEC Translocation Channels
  • Sequence Deletion
  • Signal Recognition Particle / metabolism*


  • Membrane Proteins
  • Protein Precursors
  • Protein Sorting Signals
  • SEC Translocation Channels
  • Signal Recognition Particle
  • preprolactin
  • Prolactin
  • RNA, Transfer