Plasmids used in this study

The Q-rich domains of seven different yeast proteins possess autonomous expression-enhancing (PEE) activities.

(A-B) N-terminal fusion of Rad51-NTD/SCD, Rad53-SCD1, Hop1-SCD, Sml1-NTD, Sup35-PND, Ure2-UPD and New1-NPD promotes high-level expression of LacZ-NVH, respectively. The NVH tag contains an SV40 nuclear localization signal (NLS) peptide preceding a V5 epitope tag and a hexahistidine (His6) affinity tag (27). Western blots for visualization of LacZ-NVH fusion proteins (A) and quantitative β-galactosidase assays (B) were carried out as described previously (27). Error bars indicate standard deviation between experiments (n ≥ 3). Asterisks indicate significant differences relative to wild type (WT) in A or lacking an NTD in B, with P values calculated using a two-tailed t-test (***, P value <0.001; **, P value <0.01). (C-D) The PEE activities of S/T/Q/N-rich domains are independent of the quaternary structures of target proteins. (C) Rad53-SCD1 can be used as an N-terminal fusion tag to enhance production of four different target proteins: LacZ-NVH, GST-NVH, GSTnd-NVH and GFP-NVH. (D) Visualization of native Rad51 (NTD-Rad51-ΔN), Rad51-ΔN, and the Rad51-ΔN fusion proteins by immunoblotting. Hsp104 was used as a loading control. Size in kilodaltons of standard protein markers is labeled to the left of the blots. The black arrowhead indicates the protein band of Rad51-ΔN. (E) MMS sensitivity. Spot assay showing five-fold serial dilutions of indicated strains grown on YPD plates with or without MMS at the indicated concentrations (w/v).

S. cerevisiae strains used in this study

The autonomous protein-expression-enhancing function of Rad51-NTD is unlikely to be controlled during transcription or simply arise from plasmid copy number differences.

The effects of WT and mutant Rad51-NTD on β-galactosidase activities (A), plasmid DNA copy numbers (B), relative steady-state levels of LacZ-NVH mRNA normalized to ACT1 (actin) mRNA (C), and relative ratios of LacZ-NVH mRNA versus plasmid DNA copy number (D). The wild-type yeast cells were transformed with indicated CEN-ARS plasmids, respectively, to express WT and mutant Rad51-NTD-LacZ-NVH fusion proteins or LacZ-NVH alone under the control of the native RAD51 gene promoter (PRAD51). The relative quantification (RQ = 2-ΔΔϹT) values were determined to reveal the plasmid DNA copy number and steady-state levels of LacZ-NVH mRNA by g-qPCR and RT-qPCR, respectively. LacZ and ACT1 were selected as target and reference protein-encoding genes, respectively, in both g-qPCR and RT-qPCR. The data shown represent mean ± SD from three independent biological data-points.

The oligonucleotide primers used for g-qPCR and RT-qPCR

The expression-promoting function of Rad51-NTD is controlled during protein translation and does not affect ubiquitin-mediated protein degradation.

(A) The steady-state protein levels of Rad51-NTD-LacZ-NVH and LacZ-NVH in WT and six protein homeostasis gene knockout mutants. (B-D) The impact of six protein homeostasis genes on the β-galactosidase activity ratios of Rad51-NTD-LacZ-NVH to LacZ-NVH in WT and the six gene knockout mutants (B). The β-galactosidase activities of LacZ-NVH (C) and Rad51-NTD-LacZ-NVH (D) in WT and the six gene knockout mutants are shown. Asterisks indicate significant differences, with P values calculated using a two-tailed t-test (***, P value <0.001; **, P value <0.01; *, P value <0.05).

Relative

β-galactosidase (LacZ) activities are correlated with the percentage STQ or STQN amino acid content of three Q-rich motifs. (A) List of N-terminal tags with their respective length, numbers of S/T/Q/N amino acids, overall STQ or STQN percentages, and relative β-galactosidase activities. (B-D) Linear regressions between relative β-galactosidase activities and overall STQ or STQN percentages for Rad51-NTD (B), Rad53-SCD1 (C) and Sup35-PND (D). The coefficients of determination (R2) are indicated for each simple linear regression. (E) The amino acid sequences of wild-type and mutant Rad51-NTD, Rad51-SCD1 and Sup35-PND, respectively.

Alanine scanning mutagenesis of IDRs. The amino acid sequences of WT and mutant IDRs are listed in Table S1. Total protein lysates prepared from yeast cells expressing Rad51-NTD-LacZ-NVH (A), Sup35-PND-LacZ-NVH (B) or Rad53-SCD1-LacZ-NVH (C) were visualized by immunoblotting with anti-V5 antisera. Hsp104 was used as a loading control. Quantitative yeast β-galactosidase (LacZ) assays were carried out as described in Figure 1. Error bars indicate standard deviation between experiments (n = 3). Asterisks indicate significant differences when compared to LacZ-NVH with p values calculated with a two-tailed t-test (**, P value <0.01 and ***, P value <0.001).

Percentages of proteins with different numbers of SCDs, and polyQ, polyQ/N or polyN tracts in 37 different eukaryotes.

Usage frequency (%) of standard codons [stop codon (*), Q, C, Y and W] and reassigned stop codons (→ Q, → C or → W) in 37 different eukaryotes

The numbers of SCD proteins involved in different biological processes of six representative eukaryotes

The numbers of polyQ/N proteins involved in different biological processes of six representative eukaryotes

The numbers of polyQ proteins of different biological processes in six ciliate species

Proteome-wide contents of 20 different amino acids in 37 different eukaryotes.

Percentages of proteins with indicated polyQ and polyQ/N tracts in 37 different eukaryotes.

Percentages of proteins with indicated polyX motifs in 37 different eukaryotes.