Presentation Information
[3ASBA-10]Exploring the design principles of proteins and cells through protein overexpression experiments using yeast as a model system
○Hisao Moriya1 (1. Okayama University (Japan))
Keywords:
Overexpression,Yeast,Evolution,Protein design
[Purpose] To elucidate the design principles of proteins and cellular systems by quantitatively characterizing the limits of protein overexpression and the resulting cellular responses.
[Method] Protein expression levels were systematically increased in yeast using the genetic tug-of-war (gTOW) method. For each protein, the maximal tolerated expression level (expression limit) was measured, and associated phenotypes—including growth defects, localization changes, aggregation, and organelle morphology—were quantitatively analyzed. These data were integrated with protein features to infer underlying constraint mechanisms.
[Results] Expression limits varied over a wide range across proteins and were strongly influenced by subcellular localization and protein properties. Overexpression beyond these limits induced diverse phenotypic changes, including organelle enlargement, aggregation, and emergence of abnormal cellular structures. These effects were not adequately explained by pathway-based models but were consistent with multiple constraint mechanisms such as resource overload, stoichiometric imbalance, and non-specific interactions.
[Consideration] The observed variability in expression limits suggests that cellular robustness is governed by a structured set of constraints rather than uniform buffering capacity. These constraints operate in a context-dependent and spatially organized manner, indicating that cellular systems are better understood as constrained state spaces rather than linear pathways. Overexpression serves as a perturbation that reveals these hidden constraints.
[Conclusion] Protein overexpression experiments reveal that the behavior and limits of cellular systems are determined by underlying constraint structures. This framework provides a shift from pathway-centric interpretations toward a constraint-based understanding of biological systems and offers a basis for predicting cellular responses under extreme or untested conditions.
[Method] Protein expression levels were systematically increased in yeast using the genetic tug-of-war (gTOW) method. For each protein, the maximal tolerated expression level (expression limit) was measured, and associated phenotypes—including growth defects, localization changes, aggregation, and organelle morphology—were quantitatively analyzed. These data were integrated with protein features to infer underlying constraint mechanisms.
[Results] Expression limits varied over a wide range across proteins and were strongly influenced by subcellular localization and protein properties. Overexpression beyond these limits induced diverse phenotypic changes, including organelle enlargement, aggregation, and emergence of abnormal cellular structures. These effects were not adequately explained by pathway-based models but were consistent with multiple constraint mechanisms such as resource overload, stoichiometric imbalance, and non-specific interactions.
[Consideration] The observed variability in expression limits suggests that cellular robustness is governed by a structured set of constraints rather than uniform buffering capacity. These constraints operate in a context-dependent and spatially organized manner, indicating that cellular systems are better understood as constrained state spaces rather than linear pathways. Overexpression serves as a perturbation that reveals these hidden constraints.
[Conclusion] Protein overexpression experiments reveal that the behavior and limits of cellular systems are determined by underlying constraint structures. This framework provides a shift from pathway-centric interpretations toward a constraint-based understanding of biological systems and offers a basis for predicting cellular responses under extreme or untested conditions.
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