Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response

Abstract

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components and increased functional output, and are required for survival. Remarkably, cells also showed a down-regulation of ribosomes at the protein level. That this represented an adaptive response and not an adverse toxic effect was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

Keywords: autophagy; proteasome; proteostasis; ribosome; stress response.

FIGURE 1.

Proteomic analysis of arsenic-induced toxicity. A, experimental design of the proteomic analysis of arsenic mediated toxicity. Triplicate wild-type cultures were treated with sodium arsenite (1 mm) and sampled at 0, 1, and 4 h. Extracts were prepared, standardized by protein amount, proteolytically digested, labeled with TMT probes, and subject to mass spectrometric analysis by LC-MS/MS. BPRP refers to basic pH reversed-phase chromatography. B, growth of wild-type cells during treatment with sodium arsenite (1 mm) and after drug wash-out, as indicated. C, major cellular pathways showing increased or decreased protein abundance after arsenic treatment. The numbers of co-regulated pathway components are listed in parentheses.

FIGURE 2.

Up-regulation of the proteasome by arsenic. Relative protein abundance of selected proteasome pathway components as determined by proteomic analysis at 0, 1, and 4 h after treatment with sodium arsenite (1 mm): proteasome master regulator and transcription factor, Rpn4 (panel A), integral proteasome components Pre2 (panel B) and Rpn9 (panel C), and proteasome-associated protein, Cuz1 (panel D). A control protein, Tub1, showed no change (panel E). Error bars represent S.D. from triplicate cultures. In addition, all differences between untreated and treated samples were statistically significant by Student's t test (p < 0.01), with the exception of those pertaining to Tub1.

FIGURE 3.

Up-regulation of autophagy by arsenic. A–D, relative protein abundance of selected autophagy pathway components as determined by proteomic analysis at 0, 1, and 4 h after treatment with sodium arsenite (1 mm). Panel A, Atg8; panel B, Atg19; panel C, Atg33. Act1 serves as a control (panel D). Error bars represent S.D. from triplicate cultures. In addition, all differences between untreated and treated samples were statistically significant by Student's t test (p < 0.01), with the exception of those pertaining to Act1. E, induction of autophagy in vivo, as measured by generation of free GFP from a GFP-Atg8 fusion, after treatment with sodium arsenite (1 mm). Upper panel, immunoblot with anti-GFP antibody; lower panel, anti-Pgk1 antibody (loading control). F, growth of wild-type, atg8Δ, rpn4Δ, and atg8 rpn4Δ mutants in the presence or absence of sodium arsenite, as indicated. Plates were cultured at 30 °C for 2–5 days.

FIGURE 4.

Down-regulation of ribosomal proteins by arsenic. A–D, relative protein abundance of selected ribosome subunits as determined by proteomic analysis at 0, 1, and 4 h after treatment with sodium arsenite (1 mm). Ribosome large subunit proteins Rpl35b (panel A) and Rpl21b (panel B) are shown as well as small subunit proteins Rps16b (panel C) and Rps29b (panel D). Error bars represent S.D. from triplicate cultures. In addition, all differences between untreated and treated samples were statistically significant by Student's t test (p < 0.01). E, left panel, clustering diagram showing relative protein abundance of ribosome subunits in triplicate at 0, 1, and 4 h after treatment with sodium arsenite (1 mm), as indicated. Right panel, distribution of ribosome subunits by change in protein levels after arsenic treatment. F and G, conventional immunoblot analysis for two ribosomal proteins, Rpl16a-TAP (panel F) and Rpl31b-TAP (panel G), after treatment with sodium arsenite (1 mm) for 1 h. Upper panels, anti-TAP antibody; lower panels, anti-Pgk1 antibody (loading controls).

FIGURE 5.

Ribosome reduction protects against arsenic-induced toxicity. A, growth of wild-type yeast and five otherwise unrelated ribosome mutants (rpl20aΔ, rpl39Δ, rpl19bΔ, rps17aΔ, and rps16bΔ) in the presence or absence of sodium arsenite (1.5 mm) as indicated. Plates were cultured at 30 °C for 3 days. B, growth in liquid media (YPD) of wild-type and rpl19bΔ in the presence or absence of sodium arsenite (1 mm) as indicated. Error bars represent S.D. from two independent cultures. Error bars do not overlap at the 2-, 4-, 6-, and 8-h time points in the no drug panel and at the 4-, 6-, 8-, and 10-h time points in the sodium arsenite panel. C and D, ribosome protein levels after wash-out of sodium arsenite for two subunits, Rpl16a-TAP (panel C) and Rpl31b-TAP (panel D). Upper panels, anti-TAP antibody; lower panels, anti-Pgk1 antibody (loading controls). Treatment was with 1 mm sodium arsenite for 4 h before wash-out.

FIGURE 6.

A ribosome biogenesis mutant protects against arsenic-induced toxicity. A, relative protein abundance of Slx9 as determined by proteomic analysis at 0, 1, and 4 h after treatment with sodium arsenite (1 mm). Error bars represent S.D. from triplicate cultures. In addition, the differences between untreated and treated samples were statistically significant by Student's t test (p < 0.01). B, growth of wild-type and slx9Δ strains in the presence or absence of sodium arsenite (2.0 mm) as indicated. Plates were cultured at 30 °C for 2–5 days.

FIGURE 7.

Ribosome reduction is part of a broad stress response. A, ribosomal protein abundance after treatment with sodium arsenite (1 mm) or cadmium chloride (200 μm) for 1.5 h, as visualized by immunoblot analysis for ribosomal protein Rpl16a-TAP. Upper panel, anti-TAP antibody; lower panel, anti-Pgk1 antibody (loading control). Ponceau S staining of the immunoblot also confirmed comparable loading (data not shown). B, phosphorylation of translation initiation factor eIF2-α (Ser-51) after treatment with sodium arsenite (1 mm) for the indicated times. Upper panel, anti-phospho-eIF2-α antibody; lower panel, anti-Pgk1 antibody (loading control).