Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation

Abstract

Cotranslational protein degradation plays an important role in protein quality control and proteostasis. Although ubiquitylation has been suggested to signal cotranslational degradation of nascent polypeptides, cotranslational ubiquitylation occurs at a low level, suggesting the existence of an alternative route for delivery of nascent polypeptides to the proteasome. Here we report that the nuclear import factor Srp1 (also known as importin α or karyopherin α) is required for ubiquitin-independent cotranslational degradation of the transcription factor Rpn4. We further demonstrate that cotranslational protein degradation is generally impaired in the srp1-49 mutant. Srp1 binds nascent polypeptides emerging from the ribosome. The association of proteasomes with polysomes is weakened in srp1-49. The interaction between Srp1 and the proteasome is mediated by Sts1, a multicopy suppressor of srp1-49. The srp1-49 and sts1-2 mutants are hypersensitive to stressors that promote protein misfolding, underscoring the physiological function of Srp1 and Sts1 in degradation of misfolded nascent polypeptides. This study unveils a previously unknown role for Srp1 and Sts1 in cotranslational protein degradation and suggests a novel model whereby Srp1 and Sts1 cooperate to couple proteasomes to ribosome-bound nascent polypeptides.

Keywords: Cotranslational Protein Degradation; Nuclear Import Factor Srp1; Proteasome; Protein Degradation; Protein Dynamics; Protein Processing; Protein Turnover; Ubiquitin-independent Degradation.

FIGURE 1.

Srp1 binds to the N-terminal domain of Rpn4. A, yeast two-hybrid assay showing the interaction between Srp1 and Rpn4. The isolated Srp1 clone (GAL4AD-Srp_165–542) from two-hybrid screening interacted with Rpn4_1–151-GAL4DB but not Rpn4_11–151-GAL4DB. B, Rpn4 but not Rpn4_Δ1–10 was pulled down by GST-Srp1. C, Srp1 was retained by GST fusion with Rpn4_1–229 but not Rpn4_11–229. C-terminally FLAG-tagged Rpn4 and Rpn4_Δ1–10 and N-terminally his-tagged Srp1 were expressed in E. coli cells. Input of GST fusion proteins in the pulldown assays was examined by Coomassie Blue staining (B and C, bottom panels).

FIGURE 2.

Ub-independent cotranslational degradation of Rpn4 is impaired in srp1–49. A, pulse-chase analysis for the degradation of newly synthesized Rpn4_Δ211–229-3HA expressed from the native RPN4 locus in WT and srp1–49 cells. Cells were labeled with [³⁵S]Met/Cys for 5 min and chased for different intervals as indicated. Cell extracts were subjected to IP with an anti-ha antibody, followed by SDS-PAGE and autoradiography. Rpn4_Δ211–229-3HA is marked by an arrow. B, decay curves of Rpn4_Δ211–229-3HA. ³⁵S-labeled Rpn4_Δ211–229-3HA from A was quantified by a PhosphorImager. Remaining ³⁵S-Rpn4_Δ211–229-3HA at each time point was plotted as a percentage of that at time 0. Data are mean ± S.D. of three independent experiments. C, pulse-chase analysis was carried out as in A, except that Rpn4_Δ211–229-3HA was expressed from the copper-induced CUP1 promoter on a low-copy vector. D, quantification of ³⁵S-labeled Rpn4_Δ211–229-3HA from C to show the decay curves. E, comparison of protein synthesis in WT and srp1–49 strains. Aliquots of cells were withdrawn at different time points after addition of [³⁵S]Met/Cys and used to prepare extracts. The incorporation of ³⁵S into polypeptides was measured by TCA precipitation assay. Shown are the results of three independent experiments. F, pulse-chase analysis was performed as in A, with the pulse labeling time shortened to 1 min, followed by a chase for 0 and 5 min. G, quantification of ³⁵S-labeled Rpn4_Δ211–229-3HA from F.

FIGURE 3.

Cotranslational protein degradation is impaired in srp1–49. A, bulk degradation of newly synthesized proteins is slower in srp1–49 than in WT cells. Cells were labeled with [³⁵S]Met/Cys for 1 min and chased for 0, 5, 10, 20, and 40 min. Remaining ³⁵S-labeled proteins were measured by TCA precipitation assay. B, measurement of cotranslational protein degradation. WT and srp1–49 cells deleted of PDR5 were treated with MG132 or DMSO for 10 min prior to 1-min pulse labeling with [³⁵S]Met/Cys. RNCs were separated from the supernatant (Sup) by ultracentrifugation through a 25% sucrose cushion. ³⁵S-labeled proteins in the RNC and supernatant fractions were quantified by TCA precipitation assay. Data are mean ± S.D. of three independent experiments. C, autoradiogram of RNCs following SDS-PAGE. Equal amounts of RNCs prepared as in B were applied to SDS-PAGE.

FIGURE 4.

The binding of Srp1 to ribosome-bound Rpn4 nascent chains. A, the production of ribosome-bound Rpn4_1–415 and Rpn4_80–415 polypeptides. Stop codon-less DNA templates encoding Rpn4_1–415 (long temp) and Rpn4_80–415 (short temp) were applied to the TnT coupled transcription/translation system supplemented with [³⁵S]Met/Cys. A reaction without template DNA (no temp) was used as a control. After the reactions were terminated, the mixtures were ultracentrifuged through a 0.5 m sucrose cushion. Pelleted RNCs containing ³⁵S-labeled Rpn4_1–415 or Rpn4_80–415 and unloaded ribosomes were dissolved in sample buffer and resolved by SDS-PAGE. B, measurement of the binding of Srp1 to ribosome-bound Rpn4 nascent chains. ³⁵S-labeled Srp1 was incubated with non-labeled RNCs bearing Rpn4_1–415 (lane 2), Rpn4_80–415 (lane 3), or unloaded ribosomes (lane 1). Free [³⁵S]Srp1 was removed by ultracentrifugation. Retained [³⁵S]Srp1 was resolved by SDS-PAGE and quantified by a PhosphorImager. C, quantification of the results from B. Data are mean ± S.D. of three independent experiments. The amount of Srp1 binding to Rpn4_1–415 RNC is set at 100%.

FIGURE 5.

Srp1 binds ribosome-bound nascent polypeptides. A, flow chart of experiments to show the binding of Srp1 to ribosome-bound nascent chains. Puro, puromycin. B, immunoblot (IB) analysis to show puromycin labeling and cross-linking of nascent chains. RNCs isolated from WT and srp1–49 cells were incubated with puromycin prior to the addition of the cross-linker DTSSP. The reaction mixture was heated at 95 °C for 5 min in sample buffer with or without DTT, followed by SDS-PAGE and immunoblotting with an anti-puromycin antibody. Puro-NCs, puromycin-labeled nascent chains; M, molecular markers. C, endogenous Srp1 and Srp1^E145K are physically associated with RNCs. The same samples as in B were analyzed by immunoblotting with an anti-Srp1 antibody. D and E, Srp1 binds ribosome-bound nascent polypeptides. Puromycin-labeled, DTSSP-cross-linked RNCs were disassembled by incubation with 1% SDS at 95 °C for 5 min. The samples were then diluted to reduce the SDS concentration and subjected to IP with anti-puromycin (D) or anti-Srp1 (E) antibody. The precipitates were resolved by SDS-PAGE under reducing conditions and analyzed by immunoblotting with anti-Srp1 (D) or anti-puromycin (E) antibody.

FIGURE 6.

Srp1 and Sts1 couple proteasomes to polysomes. A, polysome profiles. Cell extracts of various strains were run in parallel on 10–50% sucrose gradient ultracentrifugation. Polysome profiles were determined by plotting 254-nm absorbance against fractions collected from the top to the bottom of the gradient. B, cosedimentation of proteasomes with polysomes in WT, srp1–49, and sts1–2 cells. Fractions containing polysomes were subjected to SDS-PAGE and analyzed by immunoblotting with antibodies against Rpn12, L3, and Srp1. C, quantification of the data from A to compare the ratio of Rpn12 versus L3 in different strains. D, analysis of the binding of Sts1 to Srp1 and Srp1^E145K. N-terminally His-tagged Sts1 expressed in and purified from E. coli was applied to pulldown assays with GST-Srp1 (lane 2), GST-Srp1^E145K (lane 3), or GST (lane 4). Retained His-Sts1 was detected by immunoblotting with an anti-His antibody. E, Sts1 links Srp1 to the proteasome. Purified yeast 26 S proteasomes carrying a His-tagged Pre1 subunit were incubated with GST, GST-Srp1, or GST-Srp1^E145K in the absence (lanes 1–3) or presence (lanes 5–7) of His-Sts1. Pre1-His and His-Sts1 were detected by immunoblotting with an anti-His antibody.

FIGURE 7.

srp1–49 and sts1–2 are hypersensitive to stressors inducing protein misfolding. Five-fold serial dilutions of exponentially growing WT, srp1–49, and sts1–2 cell cultures were spotted on SD plates supplemented with essential amino acids plus CHX (0. 5 μm), hygromycin B (1 mm), or L-azetidine-2-carboxylic acid (AZC) (0.1 mm) and incubated at 30 °C for 3 days.