SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites

Abstract

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.

Figure 1. Amino acid residues 96-100 in SRP14 are critical for elongation arrest activity of SRP

(A) Sequence alignment of the C-terminal portions of wild type and mutated human SRP14 proteins (h14). (B) and (C) Elongation arrest (black) and translocation (gray) activities of SRPs (RCs) reconstituted with different h14 proteins. The right panels: quantification of the results (n≥3). EKRM: 0.2 eq./reaction. Activities were normalized to RCwt (100%.) The elongation arrest and the translocation activities of RCwt were 72±7% and 79±10%, respectively. (D) Targeting of pPL86-RNCs to microsomes. Targeting efficiencies (T [%]) were monitored by sedimenting microsomes through a sucrose cushion. EKRM: 1 eq./reaction; S: Supernatant, P: Pellet. Right panel: Quantification of the assays (n=3). T [%] was normalized to RCwt (100%). –RC: Buffer. RC(-14): SRP without h9/14. EKRM: SRP-depleted microsomes.

Figure 2. G14A12 and G14A5 fail to complement h14-depleted cells for efficient protein secretion and membrane protein accumulation

(A) SEAP activity in medium of 293T cells collected between 144-168 hr and standardized to the amount of protein present in extracts prepared from the secreting cells. shRNA(Luc)/G14: Activities were normalized to the one of mock-depleted cells expressing G14 (n=3). (B) HeLa cells stained with antibodies for cell surface expression of VSV-G and TfnR in the absence of detergent. (C) and (D) Surface staining intensities of HeLa cells expressing the GFP-h14 protein as indicated (n=2) were quantified for 200 cells each and normalized to the one of G14/shRNA(Luc). (E) Cell surface expression of glycoproteins revealed in HeLa cells stained with GS-II lectin. (F) Quantification of cell surface glycoprotein staining in G14A5 cells normalized to cells complemented with G14 (n=2).

Figure 3. Elongation arrest-defective SRP causes a defect in preprotein translocation

(A) Preprolactin (pPL3f) and prolactin (PL3f) pulse-labeled with [³⁵S]methionine and immunoprecipitated with anti-flag antibodies. T: translocation efficiency. (B) Preprolactin (pPL3f) and prolactin (PL-3f and *PL3f) accumulation revealed with anti-flag antibodies. *PL: Phosphorylated prolactin in transit for secretion. *PL3f/SUP: relative levels of *PL in the medium. (C) Same as (A) with pSEAP3f. 3fSEAP and p3fSEAP were precipitated with antibodies against SEAP (upper panel). p3fSEAP was precipitated with flag-tag antibodies (lower panel). (D) TfnR-GFP was revealed with anti-GFP antibodies. preTfnR-GFP most likely represents the preprotein. *TfnR-GFP: modified mature protein. The preTfnR-GFP values were standardized to actin and normalized to shRNA(Luc)/G14 (n=2). All experiments were done in 293T cells and equal amounts of cell extracts were loaded in each lane for Western blot analysis.

Figure 4. The absence of the elongation arrest function impairs cell growth

(A) 293T cells were plated at equal densities and live cells counted at the times indicated (n=3). h14 depletion starts at 120 hr (Lakkaraju et al., 2007). No increased cell death was observed with GA12 and GA5 cells (see 0-300 AU in C). (B) The doubling time was calculated for cell counts between 120-168 hr. Doubling times of 30-34 hr represent a 50-60% increase as compared to 21 hr of the mock treated cells. (C) Propidium iodide-stained 293T cells were analyzed for cell cycle progression using FACSort. FI: Fluorescence intensity in arbitrary units [AU]. (D) Quantification of cells in different cell cycle phases (n=5). , The sum of cells in the three phases was set to 100%. p<0.05 for cells in G0/G1phase indicates statistical significance of the data at 95% confidence levels.

Figure 5. The mutant phenotypes of G14A12 and G14A5 are specifically rescued by slowing down nascent chain elongation

(A) (B) and (C) SEAP secretion from 293T cells in the presence of anisomycin (ANM), cycloheximide (CHX) or hippuristanol. For each concentration, the secretion activities were normalized to the one of shRNA(Luc)/G14 cells (n=3). The absolute activities of mock-depleted cells were decreased to 11, 16 and 15 % at 0.03 μg/ml of anisomycin, at 3 μg/ml cycloheximide and at 0.75 μM hippuristanol, respectively, as compared to control cells. (D) Preprotein accumulation in the presence of anisomycin at 168 hr. Equal amounts of cell extracts were displayed by SDS-PAGE and pPL3f revealed with anti-flag antibodies (n=2). (E), (F) and (G) Effects of antibiotics on the cellular protein synthesis rate in mock-depleted 293T cells. Cells were labeled for 15 min. with [³⁵S]methionine/cysteine, labeled proteins were acid-precipitated and quantified (n=3). In parallel experiments we found that the [³⁵S]-uptake in shRNA(Luc)-G14, shRNA(14)-G14 and shRNA(14)-G14A5 cells without antibiotics was 31'354, 33'047 and 29'149 total counts/10⁶ cells, respectively, indicating that protein synthesis was unaffected by GFP-h14A5 expression.

Figure 6. Increasing receptor levels compensate the effects of protein mutants

(A) Western blot analysis from HeLa cells co-expressing either G14 or G14A5 with one or both SRα and SRβ. b/a indicates the increase in SR subunit expression standardized to β-actin. (B) Cellular localization of SRα and SRβ. Immunofluorescent images from HeLa cells expressing G14A5 and both SR subunits labeled with anti-SRα and anti-SRβ antibodies. (C) SEAP secretion of cells co-expressing either G14A5 or, as controls, G14 or GFP and one or both SR subunits.

Figure 7. A model for SRP-mediated targeting

The elongation arrest function of SRP reduces nascent chain length and induces ribosome stacking during the targeting process to ensure highest efficacy in protein translocation at rate-limiting SR levels. At increased SR levels, more nascent chains are target successfully even in the absence of a delay in elongation (see also text). SRP: red; SRP with black cross: SRP without the elongation arrest function; SR: green.