Schematic diagrams illustrating the constraints on protein expression levels.

A) Protein expression levels are determined by demand and constraints (created by the authors, inspired by the work by Keren et al. (Keren et al., 2016). The existence of this evolutionary principle is revealed by the relationship between fitness (growth rate) and expression level when the expression level is altered, and in general, the native expression level provides the highest level of fitness for the organism (Bruggeman et al., 2020). The existence of constraints is revealed by a decrease in fitness upon overexpression (red arrow). Constraints determine the expression limit of the protein, i.e., the expression level at which growth inhibition occurs (dotted lines), but there can be multiple constraints for a single protein. The constraint that exists at the highest expression limit is protein burden.

B) Four major mechanisms that constrain protein expression levels. The figure outlines the fate of proteins and shows what adverse effects occur upon overexpression (red arrows). The resource overload that occurs within the synthesis process (transcription and translation) is specifically referred to as protein burden. This diagram is a more concise redrawing of the author’s earlier one (Moriya, 2015). That asterisk means differentially localized proteins.

C) A barrel model for resource overload (modified from the author’s earlier one) (Kintaka et al., 2020).

The size of the barrels represents the capacity of each process, and the arrows represent the fate of the proteins processed there. The expression limit of a protein is defined by the lowest-capacity process that processes the protein. Among these processes, synthesis, which processes all proteins, is considered to have the largest capacity. Thus, proteins processed only by synthesis would have the highest expression limits, and their overexpression would overload synthesis (i.e., cause protein burden). Previous studies by the authors, conducted in this context, estimated that protein burden occurs at more than 15% of total protein (Eguchi et al., 2018).

D) Difference in burden due to gene structure (codon optimization). The amount of transcription required to achieve the same protein expression limit depends on the degree of codon optimization. This may change whether transcription or translation results in a higher burden.

E) An ideal framework for protein burden studies. See text for details.

Evaluation of expression constraint (or neutrality) of fluorescent proteins.

A) Experimental setup of the analysis. Target proteins were expressed under the control of the TDH3 promoter (TDH3pro) using the multicopy plasmid pTOW40836. The cells were pre-cultured under –Ura conditions, and then cultured in –LeuUra conditions.

B) Growth curves of the vector control and cells overexpressing FPs and their mutants in synthetic medium (–LeuUra). The solid or dotted lines show the average calculated from three biological replicates. Growth curves with the standard deviations (SD) of replicates are shown in Figure 2_S2.

C) MGR of cells overexpressing FPs and their mutants (percent over the vector control).

D) Gel images of SDS-PAGE-separated proteins extracted from cells overexpressing FPs. All proteins were separated after staining with a fluorescent dye.

E) Protein level of the target protein (percent over the total protein). The amount was calculated from the intensity of the target bands separated by SDS-PAGE of D. See Figure 2_S1B for the method of FP and total protein quantification. Note that EGFP-YG and mCherry may not be accurately measured due to dimerization and cleavage of the protein, respectively.

F) Relationship between MGR and the protein level. MGR data are the same as in C, and the protein level data are the same as in E. The neutrality indexes (NIs), calculated from the products of MGR and protein levels, are indicated by the lines. If the investigated proteins have the same NI, they are located on the same dotted line segment.

G) The neutrality index for the FPs and mutants. The neutrality index is the product of %MGR (/vector) and %protein level (/total protein).

The bars and error bars in C, E, and G show the means and SDs calculated from three biological replicates. The raw data is shown with dot plots.

Evaluation of the neutrality of Tdh3 and Gpm1.

A) Growth curves of cells with the control vector (Vector) and overexpressing Gpm1 and Tdp3 and their catalytic center mutants (CCmut) in synthetic medium (–LeuUra). The solid or dotted lines show the average calculated from three biological replicates. Growth curves with SDs of replicates are shown in Figure S3-S1.

B) MGR of cells overexpressing indicated proteins (percent over the vector control).

C) Gel images of SDS-PAGE-separated proteins extracted from cells overexpressing indicated proteins.

All proteins were separated after staining with a fluorescent dye.

D) Protein levels of the target protein (percent over the total protein). The amount was calculated from the intensity of the target bands separated by SDS-PAGE of C.

E) Relationship between MGR and the protein level. MGR data are the same as in B, and the protein level data are the same as in D. The neutrality indices (NIs) are indicated by the lines in Figure 2F.

F) The neutrality index for Gpm1, Tdh3, and their CC-mutants.

The bars and error bars in B, D, and F show the means and SDs calculated from three biological replicates. The raw data is shown with dot plots.

Transcriptional response of mox and mox-YG overexpression.

A) Comparison of transcriptional responses of mox and mox-YG overexpression. Genes that showed common or different transcriptional responses in mox and mox-YG overexpression are shown in indicated colors. Genes that showed characteristic responses are also shown. FDR: false discovery rate. r: Pearson correlation coefficient.

B) Gene groups that showed significant transcriptional changes. Among the KEGG orthology level 3 categories, gene groups in the category that were significantly up-regulated or down-regulated (FDR < 0.05) in mox or mox-YG overexpression are shown in the violin plots. Genes with significant expression changes (FDR < 0.05) within the same category are shown by swarm plots. Asterisks indicate significant differences (FDR < 0.05) between mox and mox-YG comparisons in gene groups belonging to the same category. Comparisons for all categories are shown in Figure 4_S1C.

C) Expression changes in representative amino acid, ammonium, and glucose transporters.

D) Verification of promoter activity by the reporter assay. Constructs used for promoter analysis with transcription reporters and quantitative results of RFP fluorescence values (arbitrary unit) for each promoter. Bars and error bars indicate the mean and SD of maximum fluorescence values for RFP calculated from four biological replicates. Raw data are shown as dot plots. The p-values were calculated by performing Welch’s t-test and applying Bonferroni correction. “n.s.” means p > 0.05. Detailed constructs and time series data for promoters other than those shown here are in Figure 4_S2.

Overexpression of mox-YG causes a proteome response like that of TORC1 inactivation.

A) Ratio of the total protein amount over the vector. The colored area indicates the amount of overexpressed protein. The amount was calculated from the intensity of the target bands separated by SDS-PAGE. Bars and error bars indicate the mean and SD of protein levels calculated from three biological replicates. The p-values were calculated by performing Welch’s t-test and applying Bonferroni correction. “n.s.” means p > 0.05.

B) Visualization of the proteome of the vector control and mox-YG overexpressing cells using Proteomaps (Liebermeister et al., 2014). Each polygon represents the mass fraction of the protein within the proteome. Similar colors indicate similar or closely related pathways and proteins.

C) Violin plots showing groups of genes in the KEGG orthology level 3 category whose expression was increased or decreased by mox-YG overexpression. Gene groups with significantly altered expression (FDR < 0.05) in mox-YG overexpression are shown in red. Genes with significant expression changes (FDR < 0.05) within the same category are shown by swarm plots. “+” and “–” indicate the categories with significantly increased or decreased expression, respectively, when separated into Rapamycin-responsive (Rap) and Rapamycin-non-responsive (Non-Rap) gene populations. Comparisons for all categories are shown in Figures 5_S3 and S4.

D) Volcano plots showing genes whose expression levels were altered by rapamycin treatment in the proteome results of mox-YG overexpressing cells.

E) Venn diagram representing the overlap of the proteome during mox-YG overexpression and rapamycin treatment. The number of genes in each area is shown.

For C, D, and E, published data (Gowans et al., 2018) was used for comparison with the rapamycin response genes.

mox-YG overexpression causes abnormal nucleolus formation.

A. 3D structure of the ribosome. PDB model 6GQV (Pellegrino et al., 2018) is used for the base model and colored using ChimeraX software (Meng et al., 2023). Ribosomal proteins with increased expression upon mox-YG overexpression are shown with the named structures containing them. See also Figure 6_S1 for the quantitative data.

B) Electron microscope images of the vector control and mox-YG overexpressing cells. The arrow in the image points to the nucleolus structure. N: nucleus. Images of other observed cells are shown in Figure S6_S3.

C, D) Model diagrams showing the hypothetical situation of the wild type (C) and mox-YG overexpressing cells (D). In WT cells, enough ribosomal proteins (RPs) are produced, resulting in RP-assembled rRNAs and the formation of nucleolus with normal morphology. On the other hand, in mox-YG overexpressing cells, the amount of RP is reduced due to translation competition, increasing misassembled rRNA. As a result, degradation of rRNA by exosomes may be accelerated, resulting in abnormal nucleolus morphology.

E) Growth curves of WT and mtr4-1 mutant cells with the control vector or upon overexpression of mox-YG at 30°C. The solid or dotted lines show the average calculated from three biological replicates. Growth curves with SDs of replicates are shown in Figure 6_S5.

F) Ratio of total protein levels of WT and mtr4-1 strains with the vector or upon mox-YG overexpression, calculated the total protein level of WT cells with the control vector as 100%. Gray bars indicate expression levels of proteins other than mox-YG; green shaded bars indicate mox-YG expression levels. Error bars were SDs calculated from three biological replicates.

G) Fluorescence microscopy image of nucleolus-localized protein Nsr1-mScarlet-I of the WT and mtr4-1 mutant cells with the vector or mox-YG overexpression. Bright field and merged images, and quantification of the nucleolus size are shown in Figures 6_S5D and S5E.

H) Growth curves of the cells with the vector or under mox-YG overexpression cultivated at 30°C or 38°C. The solid or dotted lines show the average calculated from three biological replicates. Growth curves with SDs of replicates are shown in Figure 6_S6A.

I, J) Final OD of the cell culture with or without 1M sorbitol at 30°C (I) or 38°C (J). The bars and error bars show the means and SDs calculated from three biological replicates. The red dotted line indicates the final OD estimated from the product of (mox-YG without sorbitol) / (Vector without sorbitol) and (Vector with sorbitol) / (Vector without sorbitol). Growth curves with SDs of replicates are shown in Figures 6_S6C, D.