Massive expression of cysteine-containing proteins is cytotoxic, manifesting as abnormal morphology in yeast cells

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing each of five fluorescent proteins harboring different properties, we revealed that cysteine content was associated with the cell elongation phenotype. We further confirmed this finding by mutational analysis of an EGFP with additional cysteines, a cysteine-containing glycolytic enzyme Tpi1, and a cysteine-less glycolytic enzyme Gpm1. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. The morphological abnormality seemed to be triggered by the S-S bond-triggered protein aggregation, and by the interaction with a chaperone Ssa1 and the heat shock transcription factor Hsf1. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins. A multicopy plasmid, pTOW40836, was trasnsformed into diploid yeast cells BY4743. The eGFP and moxGFP on the plasmid are expressed from the THD3 promoter. The cells transfected with the empty vector were used as a control and compared with the transcriptome of cells in which eGFP and moxGFP are expressed cells.