Pre-emptive Quality Control of a Misfolded Membrane Protein by Ribosome-Driven Effects

Abstract

Cells possess multiple mechanisms that protect against the accumulation of toxic aggregation-prone proteins. Here, we identify a pre-emptive pathway that reduces synthesis of membrane proteins that have failed to properly assemble in the endoplasmic reticulum (ER). We show that loss of the ER membrane complex (EMC) or mutation of the Sec61 translocon causes reduced synthesis of misfolded forms of the yeast ABC transporter Yor1. Synthesis defects are rescued by various ribosomal mutations, as well as by reducing cellular ribosome abundance. Genetic and biochemical evidence point to a ribosome-associated quality-control pathway triggered by ribosome collisions when membrane domain insertion and/or folding fails. In support of this model, translation initiation also contributes to synthesis defects, likely by modulating ribosome abundance on the message. Examination of translation efficiency across the yeast membrane proteome revealed that polytopic membrane proteins have relatively low ribosome abundance, providing evidence for translational tuning to balance protein synthesis and folding. We propose that by modulating translation rates of poorly folded proteins, cells can pre-emptively protect themselves from potentially toxic aberrant transmembrane proteins.

Keywords: endoplasmic reticulum; protein folding; protein quality control; ribosome-associated quality control; translational tuning.

Graphical abstract

Figure 1

Biosynthetic Defects in Yor1-ΔF upon Loss of EMC Function (A) Yor1-ΔF(HA) was immunoprecipitated after metabolic labeling of WT and emc7Δ cells for the indicated times. The emc7Δ strain showed reduced incorporation for Yor1-ΔF(HA) over all time points. n = 22 (WT) and 16 (emc7Δ); error bars depict SEM. (B) Radiolabeled Yor1-ΔF(HA) was immunoprecipitated from the deletion strains indicated; loss of EMC2 and/or EMC6 resulted in reduced incorporation at t = 10 in relation to WT. (C) Yor1-ΔF(HA), Gap1, and Sec22 were immunoprecipitated from WT and emc7Δ strains after metabolic labeling for the indicated times; only Yor1-ΔF showed reduced incorporation at t = 10 in relation to WT. (D) Synthesis of misfolded Ycf1 and Ste6 was quantified in WT and emc7Δ strains revealing no effect of loss of Emc7. Statistical analyses used an unpaired Student’s t test; error bars depict SD (B), (C), and (D). See also Figure S1.

Figure 2

EMC-Dependent Synthesis Defects Correlate with Specific Sites of Yor1 Misfolding (A) Cartoon of Yor1, showing relevant folding and trafficking mutations. ICL, intracellular loop; NBD, nucleotide binding domain. (B) Serial dilutions of yor1Δ or yor1Δ emc7Δ strains expressing the indicated alleles of YOR1 were spotted onto YPEG media with and without oligomycin. Emc7-associated oligomycin sensitivity correlated with misfolding defects that occurred early in the protein sequence. (C) Metabolic labeling of the indicated Yor1 alleles in wild-type or emc7Δ mutants was quantified at t = 5 min and normalized to WT; synthesis defects phenocopied oligomycin sensitivity. Statistical analysis was an unpaired Student’s t test; error bars depict SD.

Figure 3

Yor1-ΔF Synthesis Defects Reflect Ribosome-Associated Events (A) Degradation of Yor1-ΔF(HA) was similar in WT and emc7Δ cells after a 10 min pulse and chase times indicated; n = 2. (B) Deletion of ERAD factors RPN4 and CUE1 in the emc7Δ background did not restore labeling of Yor1-ΔF(HA) in relation to WT at t = 10 min. (C) Deletion of the ribosomal proteins indicated reversed the effect of EMC7 deletion on metabolic labeling of Yor1-ΔF(HA), normalized to WT at t = 10 min. (D) Serial dilutions of the indicated strains expressing Yor1-ΔF were spotted onto YPEG media supplemented with oligomycin. The emc7Δ strain showed enhanced oligomycin sensitivity; additional deletion of ribosomal proteins reversed this effect. (E) Oligomycin resistance of the indicated strains expressing Yor1-ΔF was assessed by serial dilution; deletion of HEL2, DOM34, and SLH1 restored partial oligomycin resistance, whereas deletion of LTN1 had no effect. (F) Yor1-ΔF(HA) synthesis was measured in the indicated strains and normalized to WT. Deletion of HEL2 restored Yor1-ΔF(HA) synthesis, whereas deletion of LTN1 had no effect. Statistical tests were unpaired Student’s t test; error bars depict SD. See also Figures S2, S3, and S4.

Figure 4

Sec61 Dysfunction Phenocopies EMC7 Deletion to Trigger Biosynthetic Defects (A) Metabolic labeling experiments revealed synthesis defects for both WT Yor1 and Yor1-ΔF in an ER-targeting mutant, sec61-R₂₇₅E/K₄₆₄E/K₄₇₀E, whereas a TMD-gating mutant, sec61-R₂₇₅E/R₄₀₆E, caused reduced synthesis only of Yor1-ΔF(HA), and a post-translational mutant, sec61-N₃₀₂L, was unaffected. Labeling was quantified at t = 5 and normalized to a SEC61⁺ strain. (B) Fluorescence microscopy of WT and mutant cells expressing Yor1-ΔF-GFP revealed ER localization in WT and sec61-R₂₇₅E/R₄₀₆E cells but punctate accumulation in the sec61-R₂₇₅E/K₄₆₄E/K₄₇₀E mutant. (C) Deletion of HEL2 restored Yor1-ΔF(HA) synthesis in the sec61-R₂₇₅E/R₄₀₆E strain. (D) Steady-state levels of Yor1-ΔF(HA) mRNA in the strains indicated were quantified by qPCR and normalized to that of actin with a standard curve. (E) Yor1-ΔF synthesis was measured in an emc7Δ sec61Δ ssh1Δ strain expressing either SEC61 or sec61- Q₁₂₉N, a permissive gating mutant, which did not reverse the synthesis defects associated with loss of Emc7. Statistical tests were unpaired Student’s t test and reflect the difference between the strains indicated and a WT strain; error bars depict SD.

Figure 5

Translation Initiation as a Point of Regulation of Yor1-ΔF Biogenesis (A) Cartoon depicting ER-engaged ribosomes (blue) synthesizing a polytopic membrane protein, with hydrophobic TMDs (red) causing transient stalls (brackets). In the absence of productive folding or EMC function, stalls trigger ribosome collisions (red spot) and RQC. (B) Yor1-ΔF(HA) synthesis was measured in emc7Δ strains where the ribosomal subunits indicated were unable to be ubiquitinated. In this experiment, incorporation was normalized to an isogenic emc7Δ strain. (C) Deletion of RRP6 rescued oligomycin sensitivity of an emc7Δ strain, consistent with ribosome abundance as a factor in EMC-mediated synthesis defects. (D) Deletion of OPI1 reversed oligomycin sensitivity of the emc7Δ strain. (E) Metabolic labeling of Yor1-ΔF(HA) in the strains indicated was quantified at t = 10 and normalized to WT. Deletion of OPI1 reversed the synthesis defect associated with EMC7 deletion. (F) Cartoon of factors that regulate initiation. Cdc33 (eIF4E) is essential in yeast and thus was not accessible to genetic analysis. Tif4631 (green) was a deletion suppressor; Eap1 (eIF4E-BP) was a deletion enhancer. (G) Deletion of EAP1 causes oligomycin sensitivity, which is reversed by additional loss of RRP6. (H) Metabolic labeling of the eap1Δ strain expressing Yor1-ΔF(HA) was quantified at t = 10 and normalized to WT (left). Steady-state mRNA levels were measured in the same strains by qPCR, with Ct values normalized to actin and the eap1Δ strain normalized to WT; each point represents a biological replicate comprising three technical replicates (right). Statistical analyses used an unpaired Student’s t test; error bars depict SD. See also Figures S3 and S5.

Figure 6

Polytopic Membrane Proteins Have Low Translation Efficiency (A) Translation efficiency (TE) was calculated from ribosome profiling data [38] and proteins separated into different categories on the basis of published classifications [39, 40]. Proteins with single TMDs and polytopic TMDs had lower TE than cytosolic proteins. Further separation of the polytopic group into few (4–6 TMDs) and many (>10 TMDs) further revealed reduced TE as TMD number increased. EMC clients within the >10 TMD set are indicated by colored circles. Select non-EMC clients (gray circles) are also indicated. (B) To rule out TE effects caused by protein abundance, we separated the polytopic group into low-, medium-, and high-abundance classes. No significant differences between protein abundance (ppm) were observed between the 4–6 and >10 TMD classes, suggesting that abundance alone cannot account for observed TE effects. (C) Separating low- and high-abundance proteins into 4–6 and >10 TMD classes; the observed reduction in TE for >10 TMD proteins was still observed. (D) To rule out length effects, analysis as described in (A) was restricted to proteins 200–2,000 amino acids long, revealing reduced TE for polytopic membrane proteins. In all cases, statistical analyses were Mann-Whitney tests, and error bars represent SD. See also Figure S6.