Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein

Abstract

The signal recognition particle (SRP) is a ubiquitous cytoplasmic ribonucleoprotein complex required for the cotranslational targeting of proteins to the endoplasmic reticulum (ER). In eukaryotes, SRP has to arrest the elongation of the nascent chains during targeting to ensure efficient translocation of the preprotein, and this function of SRP is dependent on SRP9/14. Here we present the results of a mutational study on the human protein h9/14 that identified and characterized regions and single residues essential for elongation arrest activity. Effects of the mutations were assessed both in cell-free translation/translocation assays and in cultured mammalian cells. We identified two patches of basic amino acid residues that are essential for activity, whereas the internal loop of SRP14 was found to be dispensable. One patch of important basic residues comprises the previously identified basic pentapetide KRDKK, which can be substituted by four lysines without loss of function. The other patch includes three lysines in the solvent-accessible alpha2 of h9. All essential residues are located in proximity in SRP9/14 and their basic character suggests that they serve as a positively charged platform for interactions with ribosomal RNA. In addition, they can all be lysines consistent with the hypothesis that they recognize their target(s) via electrostatic contacts, most likely with the phosphate backbone, as opposed to contacts with specific bases.

FIGURE 1.

Sequence alignments and structure of SRP9/14. (A) The C-terminal regions of SRP14 proteins from different eukaryotic species. Human 14 contains an alanine-rich tail specific of primate proteins. Box: basic pentapeptide essential for the elongation arrest activity of SRP. (B) Sequences of the internal loop in SRP14 proteins. The internal loop between β1 and β2 is a hallmark of SRP14 proteins and is absent in the structurally homologous SRP9 proteins. (C) Residues 50 to 74 in SRP9 proteins from different eukaryotic species. Black dots, conserved and solvent-exposed residues in SRP9; *, terminal amino acid residues of the truncated proteins SRP14-9-20C and -30C. (D) Structure model of h9/14 bound to Alu RNA. Alu RNA is shown in blue and h9 and h14 are shown in red and green, respectively. Side-chain atoms of K60, K61, K64, and R71 of h9, as well as R88 of h14, are highlighted as spheres. The structure does not include the peptide chains beyond 9A75 and 14K95 as well as the internal loop in h14 (dashed line, see also the text). Dict.disc, Dictyostelium discoideum; Enta.hist: Entamoeba histolytica; Tric.vagi, Trichomonas vaginalis. Color code for amino acids: blue, basic; red, acidic; green, polar; and black, hydrophobic.

FIGURE 2.

Effects of mutations in the C-terminal region of h14 on elongation arrest activity of SRP. (A) Mutated h14 proteins used for the assembly of SRP in vitro. Amino acids of interest are in black. ART: alanine-rich tail specific for primate h14. (B) Elongation arrest and translocation activities of reconstituted SRPs (RCs). RCs contain recombinant and canine SRP proteins, synthetic SRP RNA, and the h9/14 protein indicated. WT: Wild type. Final concentration of RC: 100 nM; EKRM: 0.02 eq./μL. [³⁵S]methionine-labeled translation products displayed by SDS-PAGE. (Left panels) The elongation arrest activity is monitored as the relative inhibition in the accumulation of preprolactin compared with cyclin. (Right panels) The translocation efficiency is determined by the percentage of total preprolactin processed into prolactin (see Materials and Methods). Cyc, a truncated version of cyclin; pPL, preprolactin; PL, prolactin. (C) Quantification of the results (n ≥ 3). Activities were normalized to RCWT, which was set to 100%. Calculated values are shown in Supplemental Table S1. B, Buffer; −9/14, RC without h9/14.

FIGURE 3.

Insertion of alanine residues between h14K95 and the basic pentapetide KRDKK or the four-lysine stretch in h14K4 abrogates the elongation arrest function. (A) Mutated h14 proteins used for the assembly of SRP in vitro. Amino acid residues of interest are in black. (B) Elongation arrest and translocation activities of RCs containing mutated h9/14 proteins. WT: wild type. Final concentration of RC: 100 nM; EDTA- and salt-washed membranes (EKRM): 0.02 eq./μL. [³⁵S]methionine-labeled translation products displayed by SDS-PAGE. Cyc, truncated version of cyclin; pPL, preprolactin; PL, prolactin. (C) Quantification of the results (n ≥ 3). Activities were normalized to RCWT, which was set to 100%. Calculated values are shown in Supplemental Table S1. B, Buffer; −9/14, RC without h9/14.

FIGURE 4.

Secretion and translocation efficiencies of HEK 293T cells expressing mutated versions of h14. (A) The different h14 proteins expressed as C-terminal fusions with GFP. Amino acid residues of interest are in black. (B) Relative expression levels of 7SL RNA. Cells were depleted of the endogenous h14 protein using shRNA complementary to the 3′ UTR of h14 mRNA (sh14) or mock depleted with shLuc and complemented with the expression of the protein indicated. The expression levels of 7SL RNA were quantified with RT-PCR and normalized to the one of mock-depleted cells expressing G14 (shLuc), which was set to 100% (n = 2). (C) SEAP activity in the medium of cells collected between 144 and 168 h and standardized to the amount of protein present in extracts prepared from the secreting cells. Activities were normalized to that of the mock-depleted cells expressing G14 (n = 2). Calculated values for C and D are shown in Supplemental Table S2. (D) Accumulation of preprolactin (pPL3F), prolactin (PL3F), and phosphorylated prolactin (*PL3F) in cells treated for 8 h with 10 μM MG132. Proteins were revealed with anti flag-tag antibodies. A: anisomycin was added to the medium at 0.03 μg/mL. The accumulation of the translocated protein prolactin in elongation arrest-defective cells is most likely explained by secondary effects on the secretory pathway as a result of inefficient translocation of endogenous proteins.

FIGURE 5.

SRP14 lacking the internal loop between β1 and β2 can confer elongation arrest activity to SRP. (A) Translation products displayed by SDS-PAGE. (B) Quantification of the elongation arrest and translocation activities of RCs (n ≥ 3). Activities were normalized to RCWT (100%). Calculated values are shown in Supplemental Table S1. B, buffer; −9/14, RC without h9/14. [RC]_final, 100 nM; EKRM, 0.02 eq/μL. The differences in absolute activities between the three fully reconstituted RCs (RCWT, RC100, RCsloop) are most likely explained by differences in the reconstitution efficiencies.

FIGURE 6.

Mutations of the conserved lysine residues at positions 60, 61, 64, of SRP9 abrogate elongation arrest activity of SRP. (A) Sequence alignment of the C-terminal portions of WT and mutated h9 proteins. Sequences of interest are highlighted in black and mutated amino acid residues are highlighted in gray. (B) Elongation arrest (left panel) and translocation (right panel) activities (n ≥ 2) of particles (RCs) reconstituted with the h9 proteins indicated. B, buffer; −9/14, RC without h9/14. [RC]_final, 100 nM; EKRM, 0.02 eq/μL. Activities were normalized to RCWT (100%). Calculated values are shown in Supplemental Table S1. (C) Relative expression levels of 7SL RNA in 293T cells collected at 144 h (left panel). Expression levels of 7SL RNA were normalized to the one of mock-depleted and G9 expressing cells, which was set to 100% (n = 2). (D) SEAP activities in medium of 293T cells collected between 120 and 144 h and standardized to the amount of proteins present in extracts prepared from the secreting cells at 144 h (right panel). Activities were normalized to that of the mock-depleted cells expressing G9 (n = 2). Calculated values for C and D are shown in Supplemental Table S2. (D) Accumulation of preprolactin (pPL3F), prolactin (PL3F), and phosphorylated prolactin (*PL3F) in 293T cells after h9-depletion and complementation with the G9 proteins indicated. Prolactin was revealed with anti flag-tag antibodies.