Yeast complementation assays as a screening tool for urea, water, and ammonia permeability of membrane channels

Description

Yeast is a versatile tool for testing membrane channels in vivo. Yeast complementation assays enable the characterization of permeability and pH gating of transmembrane channels for water, urea, or ammonia permeability in liquid culture. In these assays, the functionality of exogenous proteins expressed in deletion strains is tested by observing growth or survival. Yeast cells can survive/grow under the respective assay conditions only if the exogenous protein is substituting the function of the deleted endogenous protein. To test urea permeability, a urea uptake deficient Saccharomyces cerevisiae strain that expresses the construct of choice is used. It grows in liquid media at different pHs with urea as the sole nitrogen source. The same principle applies to ammonia permeability testing with a yeast strain deficient in ammonia channels and ammonia as the only nitrogen source. For water as a solute, the samples are rapidly frozen, followed by survival determination via a fluorescent readout (FDA and PI). The expression of water-permeable channels increases the respective survival rate. The assays are established with known water channels hAQP1 and 3 and ammonia channels as AtTIP2;1 and hAQP8. Yeast complementation assays are also used as screening tools to test functional differences of membrane channels in comparison to the WT protein. We tested functional differences of various affinity tag positions on the example of HpUreI, an inner membrane pH-gated urea channel of Helicobacter pylori. Affinity tags play an integral role in protein purification. Despite several alerting indications, their influence on protein structure and function in in vitro experiments is often unknown or disregarded, which unnecessarily renders data interpretation ambiguous. Our results emphasize the need for in vivo characterization. Not only the overall tag position but also a minor amount of residual N- or C-terminal amino acids after tag cleavage, influences HpUreI's functionality. Furthermore, similar pH-independent ammonia permeabilities of all tag constructs indicate a complex solute selectivity mechanism. Yeast complementation assays are cost-effective and can be adapted for testing a broad range of solutes, studying protein homologs or mutants, and used as a pre-screening method for affinity tag positions before quantitative in vitro protein characterization.
Keywords: affinity tag position, pH-gated urea channel, Helicobacter pylori, HpUreI, yeast complementation assays, ammonia, water permeability
Supported by: P 35541 and P 31074 to AH from the Austrian Science Fund FWF

Period	26 Jun 2024
Event title	21st IUPAB Congress 2024
Event type	Conference
Location	Kyoto, JapanShow on map
Degree of Recognition	International