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A Semi-Quantitative Yeast Complementation Platform for Characterizing Urea and Ammonia Transport by Membrane Channels
Anna Stoib1,2, Sahar Shojaei1,2, Christine Siligan1
1Johannes Kepler University Linz, Institute of Biophysics, Linz, Austria.
Current Protocols
|March 6, 2026
Summary
Yeast complementation assays offer a cost-effective method to study transmembrane channel transport of urea and ammonia. This in vivo approach quantifies channel activity and pH dependency, providing valuable insights into protein function.
Area of Science:
- Molecular Biology
- Biophysics
- Microbiology
Background:
- Transmembrane channels play crucial roles in cellular transport.
- Characterizing channel permeability and pH gating is essential for understanding cellular function.
- Existing methods for studying channel transport can be limited in throughput or prone to artifacts.
Purpose of the Study:
- To detail a yeast complementation assay for quantifying urea and ammonia transport through transmembrane channels.
- To establish a method for determining solute-specific permeability and pH-dependency profiles.
- To provide a cost-effective, high-throughput alternative to existing in vitro and oocyte-based assays.
Main Methods:
- Utilizing Saccharomyces cerevisiae deletion strains in a liquid culture system.
- Employing functional complementation by monitoring cell growth on selective media with urea or ammonia as the sole nitrogen source.
- Testing bacterial urea channel HpUreI (and variants) and human aquaporin hAQP8 for transport activity across a pH range.
Main Results:
- Demonstrated the ability of the yeast assay to functionally complement deletion strains for urea and ammonia transport.
- Generated solute-specific permeability and pH-dependency profiles for tested channels.
- Showcased the assay's scalability, cost-effectiveness, and adaptability for diverse solutes and mutants.
Conclusions:
- Yeast complementation assays provide a robust, in vivo platform for characterizing transmembrane channel transport.
- This method offers advantages in throughput, cost-effectiveness, and preservation of native protein-lipid interactions compared to other techniques.
- The assay serves as an efficient tool for pre-screening channel function and guiding further in vitro studies.
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