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.

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

Fig. 1

Signal sequence is recognized by SRP and this interaction is detected by site-specific photocrosslinking. (a) Scheme of a typical signal sequence (see text for details). a. a., amino acid residues. (b) Scheme of the site-specific incorporation of photocrosslinking probe into a protein. mRNA contains amber-stop mutation (UAG) in position 18 of pPL (shown as an octagon). Premature termination of protein synthesis at this position is occurred when an amber-suppressor is absent in translation (1). However, in the presence of amber-suppressor tRNA the termination is suppressed at the amber-mutation and specific amino acid is incorporated by the amber-suppressor tRNA (Lys-tRNA^amb) in response of an amber-codon (2). When translation reaction contains modified amber-suppressor-tRNA carrying amino acid with photoreactive probe (εANB-Lys-tRNA^amb), a modified amino acid residue is incorporated into unique position of the protein that corresponds to a position of amber-mutation in the mRNA (3). Homogeneous population of RNCs is obtained by translation of mRNAs that are truncated in the coding region (for example, codon 86). Normal termination is not occurred at the end of such mRNAs because natural stop codon is missing and nascent chain is not released from the ribosome. As a result, all ribosome bound nascent chains in the reaction have the same length which is determined by the length of the truncated mRNA. (c) Photocrosslinking of nascent preprolactin (86 amino acid long nascent chain) is probe-specific and SRP-dependent. In vitro translation contained unmodified LystRNA^amb in the control reaction without photo-crosslinking probe (lane 1), εANB-Lys-tRNA^amb (lanes 2 and 3), canine SRP was added where indicated (lanes 1 and 2). [¹⁴C] molecular weight protein markers are shown in lane 4. Proteins were synthesized in vitro by wheat germ translation system in the presence of [³⁵S] methionine. See details for reaction conditions and photolysis in Material and Methods. Samples were analyzed by 10-15% gradient SDS-PAGE. The positions of nascent chains (NC), peptidyl-tRNA (Pt), and photoadduct to SRP54 (NC-SRP54) are shown. Apparent molecular masses are shown in kDa.

Fig. 2

Affect of charged amino acid residues in the N-terminal region of signal sequence (a) and in the early part of the mature protein (b) of the nascent pPL on photocrosslinking to SRP54. Stable RNCs containing the first 86 residues of either wild-type (WT) or mutant pPLs with different mutations were synthesized in vitro translation system in the presence of canine SRP. A photoreactive probe was cotranslationally incorporated into the middle of the signal sequence (position 18) as shown in Fig. 1b. Following photolysis (UV-irradiation), samples were analyzed by electrophoresis in 10-15% gradient SDS-PAGE and photoadducts were detected by phosphorimager. The quantities of mutant nascent chain-SRP54 photoadducts were calculated in per cents relatively to the photoadduct of the wild-type pPL (n=6 for 1K-c, 2K-c, 3K-c, 5D-c; n=10 for 4K-c, n-3KR-4K-c; n=12 for 5K-c; n=2 for n-3LR, n-4L; n=4 for n-3KR, n-Δ10,R; mean ± S.D.). See Table 1 for mutations’ abbreviations and their positions.

Fig. 3

Hydrophobic core of a signal sequence is a primary feature for SRP recognition. Quantitative analysis of SRP photocrosslinking to mutated pPLs with deletions in the hydrophobic core is shown in the graph (n=6, mean ± SD). Samples were analyzed as in Fig. 2. See Table 1 for mutations.

Fig. 4

Targeting and translocation of mutated pPLs across ER membrane. Mutations in the n-terminal region (a) of pPL signal sequence as well as in the early portion of mature protein (b) and hydrophobic core (c) influence targeting to translocon, translocation and signal sequence cleavage. (a-c) Full-length polypeptides of wild-type preprolactin (WT) or mutated proteins were translated in reticulocyte lysate in the absence (−) or presence (+) of rough microsomes (RM). See Table 1 for abbreviations of mutations. (d) Proteinase K protection of the proteins translocated into rough microsomes. Full-length polypeptides were translated as in figures (a) – (c) and were treated (+) or not (−) with proteinase K (PK) as indicated. Figures represent efficiency of protein translocation in per sent of the mature form (data are from 2-5 experiments; n.d., not determined). Positions of matured pPL and its precursor are marked by one and two black dots, respectively. Apparent molecular masses are shown in kDa.