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bioRxiv · 10.64898/2026.09.24.754258

Limit-pushing overexpression reveals constraints on protein abundance

Namba, S.·Nishida, Y.·Boone, C.·Andrews, B.·Moriya, H.
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Abstract

Proteins are often classified as toxic or non-toxic without measuring the abundance reached, leaving constraints on tolerable protein abundance unresolved. We established a limit-pushing approach in Saccharomyces cerevisiae combining strong inducible expression with gTOW-mediated high-copy selection to counteract copy-number compensation while measuring protein abundance and growth. Nearly all of approximately 80 chromosome I proteins severely inhibited growth or reduced viability at sufficiently high abundance. We established IE50, the expression level associated with a 50% reduction in growth rate, to quantify their widely varying overexpression tolerance. IE50 was positively associated with predicted structural order and cytoplasmic localization propensity and negatively associated with sulphur content. Single-cell imaging linked higher tolerance to proteins remaining cytoplasmic without becoming aggregation-positive and revealed abundance-dependent changes in localization and organelle morphology. At extreme abundance, Fun12, Nup60, and Pex22 generated distinct large-scale intracellular states through specific sequence regions. These findings establish overexpression toxicity as a quantitative property linked to protein characteristics and reveal both constraints on tolerable abundance and sequence-dependent capacities for intracellular organization.

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Namba, S., Nishida, Y., Boone, C., Andrews, B., Moriya, H.. 2026-09-28. Limit-pushing overexpression reveals constraints on protein abundance. https://doi.org/10.64898/2026.09.24.754258

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