Limit-pushing overexpression reveals constraints on protein abundance
- 1Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Okayama, Japan
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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BibTeX
@misc{namba2026limitpushing,
title = {{Limit-pushing overexpression reveals constraints on protein abundance}},
author = {S. Namba and Y. Nishida and C. Boone and B. Andrews and H. Moriya},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.24.754258},
url = {https://www.biorxiv.org/content/10.64898/2026.09.24.754258v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Limit-pushing overexpression reveals constraints on protein abundance
AU - S. Namba
AU - Y. Nishida
AU - C. Boone
AU - B. Andrews
AU - H. Moriya
PY - 2026
DA - 2026/09/24
PB - bioRxiv
DO - 10.64898/2026.09.24.754258
UR - https://www.biorxiv.org/content/10.64898/2026.09.24.754258v1
AB - 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.
ER -