Evaluating protein complexes between human aquaporin and calmodulin using biomolecular fluorescence complementation

Abstract

Aquaporins (AQPs) are a family of integral membrane proteins crucial for the flow of water and other small molecules across cellular membranes. The involvement of calmodulin (CaM), a multifunctional calcium-binding protein, has emerged as a central regulator for specific aquaporin homologues from eukaryotes. Using a systematic approach, applying advanced high throughput screening methods in vivo, combining flow cytometry with microscopy, we have evaluated the putative interaction between CaM and the 13 human AQP homologues recombinantly produced in the yeast Saccharomyces cerevisiae. This comprehensive approach is complemented by a theoretical validation of potential CaM binding sites and a review of confirmed CaM binding locations from previous research. Our investigation is based on the established interaction of hAQP0 and CaM and we have successfully validated the binding of hAQP1 and hAQP4 to CaM. Noteworthy, discernibly high fluorescence frequency signals were observed for hAQP8 and hAQP9, which did not correlate with a particularly high production level, supporting protein complex formation with CaM for those AQP homologues. Overall, we present a systematic approach to screen novel membrane protein interactions in vivo, relying on co-expression in yeast of Bimolecular Fluorescence Complementation (BiFC) complexes providing new insights into the regulation of the hAQPs.

Keywords: Saccharomyces cerevisiae; Aquaporin; Bimolecular fluorescence complementation; Calmodulin.

Fig. 1

Theoretical CaM binding motifs in the N- and C-terminus of the human aquaporin homologues. The Calmodulin Target Database was used to identify potential CaM binding sites in hAQP0-hAQP12. The location of the putative CaM-binding motifs is highlighted in yellow and score 0–9 is used to indicate the strength of the binding. Additionally, interaction sites to CaM, which has been confirmed by experiments, are highlighted in red.

Fig. 2

Quantification of BiFC signal through flow cytometry from yeast cells producing complexes of CaM and each of the hAQP homologues (A) The average fluorescence intensity and standard error of mean are displayed for each hAQP-CaM pair. (B) The average percentage of fluorescent cells and standard error of the mean are shown for each hAQP-CaM pair.

Fig. 3

Fluorescence and bright-field images of BiFC hAQP-CaM complexes produced in S. cerevisiae cells and images where both channels were merged. The strongest fluorescence signal was achieved from YFP_N-AQP4 + YFP_C-CaM, clearly indicating a constructive complex formation, as well as from YFP_N-AQP0 + YFP_C-CaM. Weak fluorescence was observed for the BiFC complexes showing low to intermediate fluorescence frequency. No fluorescence was observed in cells producing YFP_N-AQP6 + YFP_C-CaM, YFP_N-AQP11 + YFP_C-CaM, YFP_N-AQP12 + YFP_C-CaM as well as untransformed S. cerevisiae cells, providing a better negative control for the microscopy analysis than the BiFC formation control lacking the AQPO C-terminus, YFP_N-AQP0ΔC + YFP_C−CaM.

Fig. 4

Immunoblot analysis of the different hAQP-CaM BiFC complexes. To analyze and compare different production levels of the hAQP-CaM complexes, Immunoblot using a primary antibody towards the YFP_N-hAQP fragment was applied. Total protein, 30 µg, was loaded for each sample and even loading was confirmed by Ponceau staining of the membrane before incubation with the primary and secondary antibody, respectively. A typical Immunoblot is shown, highlighting the area of interest, and the estimated fragment size (Mw) is listed below for each hAQP-CaM complex. As is commonly observed for membrane proteins, the YFP_N-hAQP fragments migrate at a slightly lower molecular weight than the one predicted theoretically.