The ultimate overexpression of a protein could cause growth defects, which are known as the protein burden. However, the expression limit at which the protein-burden effect is triggered is still unclear. To estimate this limit, we systematically measured the overexpression limits of glycolytic proteins in Saccharomyces cerevisiae. The limits of some glycolytic proteins were up to 15% of the total cellular protein. These limits were independent of the proteins’ catalytic activities, a finding that was supported by an in silico analysis. Some proteins had low expression limits that were explained by their localization and metabolic perturbations. The codon usage should be highly optimized to trigger the protein-burden effect, even under strong transcriptional induction. The S–S-bond-connected aggregation mediated by the cysteine residues of a protein might affect its expression limit. Theoretically, only non-harmful proteins could be expressed up to the protein-burden limit. Therefore, we established a framework to distinguish proteins that are harmful and non-harmful upon overexpression.

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Extreme overproduction of gratuitous proteins can overload cellular protein production resources, leading to growth defects, a phenomenon known as the protein burden/cost effect. Genetic screening in the budding yeast Saccharomyces cerevisiae has isolated several dubious oRfs whose deletions mitigated the protein burden effect, but individual characterization thereof has yet to be delineated. We found that deletion of the YJL175W ORF yielded an N-terminal deletion of Swi3, a subunit of the SWI/SNF chromatin remodeling complex, and partial loss of function of Swi3. The deletion mutant showed a reduction in transcription of genes encoding highly expressed, secreted proteins and an overall reduction in translation. Mutations in the chromatin remodeling complex could thus mitigate the protein burden effect, likely by reallocating residual cellular resources used to overproduce proteins. This cellular state might also be related to cancer cells, as they frequently harbor mutations in the SWI/ SNF complex. Expression levels of intracellular proteins are tightly controlled to maintain organism capacity for proliferation and survival, and an excess of proteins can cause cellular dysfunction 1-3. Potentially, any harmless protein inhibits cell growth when it is extremely overproduced, because it depletes cellular protein production resources. This phenomenon is known as the protein burden/cost effect 3-6. The protein burden effect was initially observed as growth defects of bacterial cells overexpressing gratuitous proteins 7 , and later analyzed in yeast as well 2,4,6,8. The protein burden effect is triggered by the cost of gene expression upon overexpression of gratuitous proteins; the overexpression overloads cellular transcription and translation resources 4,9. Because the protein burden effect is triggered by the massive overexpression of unneeded proteins, cancer cells, where an increase in the chromosome numbers is common 10,11 , should be under the condition of the protein burden effect. While the protein burden effect initially appears to be a simple phenomenon, little is known about the physiological conditions and cellular responses triggered by the protein burden effect. Extreme overexpression of fluorescent proteins such as GFP and RFP are thought to trigger this effect 4,6,8,12. To clarify the physiology of cells suffering from protein burden effect, we recently conducted genetic profiling 13. Upon isolating a series of deletion and temperature-sensitive mutants harboring genetic interactions affecting the overproduction of GFP (GFP-op) in the budding yeast Saccharomyces cerevisiae, we found that the deletion of certain uncharacterized ORFs mitigated growth defects triggered by GFP-op. However, the molecular details underpinning alleviation of the protein burden effect in these mutants remained unclear. The SWI/SNF complex is a chromatin remodeling complex that remodels nucleosomes and changes chromatin structure by using the hydrolysis energy of ATP 14. The SWI/SNF complex is evolutionarily conserved in

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