Functional Cooperativity among the Subunits of the Homotetrameric Aquaglyceroprotein GlpF

Abstract

Aquaporins are conserved throughout all kingdoms of life. Each aquaporin monomer possesses a channel that facilitates the transport of water across the membrane, and yet the protein assembles into a tetramer. We investigated the physiological function of the tetrameric assembly by introducing destabilizing point mutations. Therefore, we overexpressed the GlpF variants in E.coli, purified them and reconstituted them into large unilamellar vesicles (LUVs) or giant (GUV) unilamellar vesicles. Subsequently, we osmotically deflated the vesicles. Our new adaptation of the Rayleigh-Gans-Debye equation served to calculate the water efflux from proteoliposomes (PL) based on the accompanying increase in scattered light intensity (Horner et al., 2015). We adopted the micropipette aspiration technique to quantify water efflux from GUVs. Reconstituted GUVs allowed direct determination of GlpF membrane concentration by fluorescence correlation spectroscopy. In order to obtain the channel abundance in LUVs, we exploited fluorescence correlation spectroscopy to count the number of particles before and after detergent-mediated vesicle solubilization (Knyazev et al., 2013). Depending on the total GlpF concentration, we found functional GlpF monomers, dimers, trimers, and tetramers. Spreading the vesicles on mica enabled us to acquire high-speed AFM imaging of the different oligomeric states. Destabilization of the tetrameric assembly was accompanied by looser packing and changes in unitary permeability. The observation suggests that tetramerization serves to control the functional state of each monomer.1. Horner et al. (2015). The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues. Science Advances 1, e1400083.2. Knyazev et al. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem. 288, 17941-17946.