A Semi‐Quantitative Yeast Complementation Platform for Characterizing Urea and Ammonia Transport by Membrane Channels

Yeast complementation assays provide a robust in vivo platform for characterizing the permeability and pH gating of transmembrane channels. This article details a liquid culture approach to quantify urea and ammonia transport using Saccharomyces cerevisiae deletion strains. Functional complementation, evidenced by cell growth in selective medium with urea or ammonia as the sole nitrogen source, directly reports on channel activity, generating solute-specific permeability and pH-dependency profiles. We present step-by-step procedures using the bacterial urea channel HpUreI of Helicobacter pylori, including two variants (A57C and L134C) for urea permeability and HpUreI, HpUreI E177Q, and human hAQP8 for ammonia transport. By monitoring growth across a pH range, this method enables semi-quantitative comparison of channel function. The assay is cost effective, scalable to high-throughput formats, and adaptable for studying diverse solutes, protein homologs, or mutants. It also serves as an efficient pre-screening tool for affinity tag placement before in vitro characterization. Unlike in vitro reconstitution, this approach preserves native protein-lipid interactions and avoids purification artifacts, allowing direct comparison to wild-type proteins. Though less quantitatively precise than in vitro methods, it offers higher throughput and solute flexibility compared to oocyte expression systems. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Quantifying urea permeability and pH gating using a yeast complementation growth assay Alternate Protocol: Adapting the yeast complementation assay to assess ammonia permeability and pH dependency.