Impact of maximal overexpression of a non-toxic protein on yeast cell physiology

While it is recognized that excess expression of non-essential proteins burdens cell growth, the physiological state of cells under such stress is largely unknown. This is because it is challenging to distinguish between adverse effects arising from the properties of the expressed excess protein (cytotoxicity) and those caused solely by protein overexpression. In this study, we attempted to identify the model protein with the lowest cytotoxicity in yeast cells by introducing a new neutrality index. We found that a non-fluorescent fluorescent protein (mox-YG) and an inactive glycolytic enzyme (Gpm1-CCmut) showed the lowest cytotoxicity. These proteins can be expressed at levels exceeding 40% of total protein while maintaining yeast growth. The transcriptome of cells expressing mox-YG to the limit indicated that the cells were in a nitrogen source requirement state. Proteome analysis revealed increased mitochondrial protein and decreased ribosome abundance, similar to the inactivated state of the TORC1 pathway. The decrease in ribosome abundance was presumably due to defective nucleolus formation, partially rescued by a mutation in the nuclear exosome. These findings suggest that massive overexpression of excess protein, termed protein burden, causes nitrogen source starvation, a metabolic shift toward more energy-efficient respiration, and a ribosomal biosynthesis defect due to an imbalance between ribosomal protein and rRNA synthesis in the nucleolus.