An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation

Abstract

The signal recognition particle (SRP)-dependent targeting pathway facilitates rapid, efficient delivery of the ribosome-nascent chain complex (RNC) to the protein translocation channel. We test whether the SRP receptor (SR) locates a vacant protein translocation channel by interacting with the yeast Sec61 and Ssh1 translocons. Surprisingly, the slow growth and cotranslational translocation defects caused by deletion of the transmembrane (TM) span of yeast SRbeta (SRbeta-DeltaTM) are exaggerated when the SSH1 gene is disrupted. Disruption of the SBH2 gene, which encodes the beta subunit of the Ssh1p complex, likewise causes a growth defect when combined with SRbeta-DeltaTM. Cotranslational translocation defects in the ssh1DeltaSRbeta-DeltaTM mutant are explained by slow and inefficient in vivo gating of translocons by RNCs. A critical function for translocation channel beta subunits in the SR-channel interaction is supported by the observation that simultaneous deletion of Sbh1p and Sbh2p causes a defect in the cotranslational targeting pathway that is similar to the translocation defect caused by deletion of either subunit of the SR.

Figure 1.

Genetic interactions between the SR and the translocons. (A) Haploid yeast strains expressing wild-type or soluble (ΔTM) forms of SRβ and wild-type or mutant alleles of Sec61 (sec61EE) or Ssh1 (ssh1EE) from plasmids as the sole source of each protein were grown to mid-log phase in SEG media at 30°C. Growth rates were compared by serial dilution analysis on YPD plates at both 30 and 37°C as described in Materials and methods. Plates were photographed after 2 (30°C) or 3 d (37°C) of growth. (B) Yeast strains (SSH1 or ssh1Δ) expressing wild-type Sec61p and SRβ-ΔTM from low-copy plasmids were transformed with high-copy plasmids (2μ) encoding SRβ-ΔTM, soluble SR (SRα + SRβ-ΔTM), or the Ssh1p complex (SSH1*; Ssh1p + Sbh2p + Sss1p). Growth rates of yeast strains on YPD media were compared as described in A. (C) Growth rates of selected yeast strains in liquid YPD media at 30°C. Plotted values are derived from a single experiment. Error bars represent SD.

Figure 2.

Cotranslational translocation defects of yeast that express SRβ-ΔTM. (A–D) Wild-type (wt) and mutant yeast cells were grown to mid-log phase at 30°C in SEG media. The cultures were diluted into SD media and allowed to grow for 4 h (A, C, and D) or 24 h (B) at 30°C. Wild-type or mutant cells (4 A₆₀₀) were collected and pulse labeled for 7 min. DPAPB immunoprecipitates was resolved by SDS-PAGE. Glycosylated (DPAPB) and nonglycosylated (pDPAPB) forms of DPAPB were quantified with a molecular imager to calculate the percent precursor. Plasmids encoding the soluble SR (SRα + SRβ-ΔTM) and the Ssh1 complex are designated as SR-ΔTM and SSH1*, respectively. (A and B) Total incorporation of Tran-³⁵S-label (pDPAPB + DPAPB) in each strain is expressed relative to the wild-type strain.

Figure 3.

Slow targeting of Dap2-RNCs in the ssh1Δ SRβ-ΔTM mutant. (A) Dap2 reporters consist of (1) the N-terminal cytoplasmic domain of Dap2p (green; Dap2p_1–29) followed by the TM span (black; Dap2p_30–45), (2) 49–265-residue spacer segments (cyan) derived from Dap2p, (3) a Ub domain (red), (4) a 42-residue linker (blue) with a processing site (arrowhead) for a Ub-specific protease, and (5) a Ura3 reporter domain followed by a triple-HA epitope tag (yellow). Sites for N-linked glycosylation (Y-shaped symbols) are indicated. (B) Cleavage of the Dap2 reporter defines the in vivo kinetics of translocon gating in the SRβ-ΔTM strain. Soluble (dark blue) and membrane-bound (light blue) pools of the SR might interact with the SRP–RNC or the translocon. Any slow step (e.g., SRP binding, SRP–RNC targeting, and RNC docking or signal insertion) that precedes translocon gating allows folding of Ub in the cytosol and cleavage of the reporter. The color code for Dap2 segments is as defined in A. (C) In vivo cleavage of the Dap2 reporter in wild-type, SRβ-ΔTM, or ssh1Δ SRβ-ΔTM strains after 4 h of growth in SD media. Labels designate the intact glycosylated (e.g., g49) and cleaved (Ura3-HA) reporter domains. Downward-pointing arrowheads designate nonglycosylated intact reporters that are diagnostic of cytosolic Dap2 reporter aggregates (Cheng et al., 2005; Cheng and Gilmore, 2006). (D) Spacer length dependence of Dap2 reporter cleavage (percent cytosolic Ura3-HA) for the wild-type (squares), ssh1Δ (circles), SRβ-ΔTM (triangles), and ssh1Δ SRβ-ΔTM (closed diamonds) yeast strains after 4 h of growth in SD media. Dap2–265 cleavage was assayed 3 h after shifting the SRβ mutant (srp102K51I) to 37°C (open diamond). (E) In vivo cleavage of the Dap2 reporters in the absence or presence of cycloheximide (CH) after 24 h of growth in SD media. Molecular weight markers for C and E are identical. (F) Spacer length dependence of Dap2 reporter cleavage (percent cytosolic Ura3-HA) for the ssh1Δ (circles), SRβ-ΔTM (triangles), or ssh1Δ SRβ-ΔTM (closed diamonds, −CH; open diamonds, +CH) yeast strains after 24 h of growth in SD media. Data points in D and F are means of two or three experiments. Color-coded error bars in D and F designate individual data points for experiments that were performed twice or SDs for experiments performed three times.

Figure 4.

Genetic interactions between translocon β subunits and SRβ-ΔTM. (A) Growth rates of ssh1Δ, sbh1Δ, or sbh2Δ yeast strains expressing the soluble SR (SRα + SRβ-ΔTM) and wild-type Sec61p were determined by serial dilution analysis at 30 or 37°C as in Fig. 1. (B) Equal amounts of total protein (50 μg/lane) were resolved by PAGE in SDS for protein immunoblot analysis using a C-terminal–specific antibody to Sec61p. The blots were also probed with antisera to 3-phosphoglycerate kinase (PGK) as a loading control. (C) Diagrams of intact Sbh2p and N-terminal deletion mutants showing the location of the transmembrane (TM) domain and the proposed cytosolic GEF domain. All Sbh2 expression constructs retain the AUG initiation codon. The segment labeled Secβ structure is homologous to the segment of M. jannaschii Secβ (residues 21–52) that was resolved in the crystal structure of SecYEβ. (D) Growth rates of the SEC61sbh2Δ SRβ-ΔTM strain after transformation with a low-copy plasmid encoding intact or C-terminal fragments of Sbh2p.

Figure 5.

Translocation defects in sbh1Δsbh2Δ mutants. (A and B) The integration of DPAPB (A) and translocation of CPY (B) were evaluated by 7-min pulse labeling of wild-type (wt) and mutant yeast strains (sbh1Δ, sbh2Δ, and sbh1Δsbh2Δ) after 4 h of growth in SD media at 30°C. The glycosylated (DPAPB) and nonglycosylated (pDPAPB) forms of DPAPB and the ER (p1CPY) and precursor (ppCPY) forms of CPY are labeled. Asterisks designate incompletely glucose-trimmed forms of glycosylated DPAPB and p1CPY. (C) Protein immunoblot detection of DPAPB-HA or CPY in wild-type and sbh1Δsbh2Δ strains. Total cell extracts were prepared for SDS-PAGE with or without prior digestion by endoglycosidase H (Endo H). Precursor and mature forms of DPAPB-HA are labeled. Deglycosylated mature CPY (dgCPY) is resolved from vacuolar CPY (m), preproCPY (p), and a hypoglycosylated form of mCPY (−1). (D) Differential centrifugation of spheroplast lysates prepared from sbh1Δsbh2Δ, srp102(K51I), and wild-type yeast strains. Supernatant (S) and pellet (P) fractions were obtained after centrifugation at 100 Kg. SRα and SRβ were detected using antisera raised against yeast SR. (E–G) Plasmids encoding full-length or N-terminal deletion alleles of Sbh2 were transformed into the sbh1Δsbh2Δ strain. (E) Growth rates of wild-type and mutant strains were compared by serial dilution analysis as described in Fig. 1. (F) Integration of DPAPB and translocation of CPY were evaluated after 24 h of growth in SD media. (G) In vivo cleavage of Dap2 reporters after 24 h of growth in SD media. Labels designate the intact glycosylated (e.g., g265), intact nonglycosylated (arrowheads), and cleaved (Ura3-HA) reporter domains. Spacer length dependence of Dap2 reporter cleavage in wild type (squares), SRα null (triangles), sbh1Δsbh2Δ (closed circles), and the sbh1Δsbh2Δ mutant expressing sbh2Δ37 (open circles). Data points are means of two experiments, one of which is shown above the graph. Data for the wild-type strain is taken from Fig. 3 D. Color-coded error bars designate individual data points for experiments that were performed twice.