Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET.

Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and robustness. We outline instrument setup and image acquisition and further describe steps for image preprocessing and two-hybrid FRET analysis using provided software to simplify the workflow. This protocol is compatible with confocal microscopes for high-precision and imaging plate readers for high-throughput applications. A plasmid-based reference system supports fast establishment of the protocol. For complete details on the use and execution of this protocol, please refer to Rivas et al.1.

Anion transport by the cochlear motor protein prestin.

Prestin is a member of the SLC26 solute carrier family and functions as a motor protein in cochlear outer hair cells. While other SLC26 homologues were demonstrated to transport a wide variety of anions, no electrogenic transport activity has been assigned so far to mammalian prestin. We here use heterologous expression in mammalian cells, patch clamp recordings and measurements of expression levels of individual cells to study anion transport by rat prestin. We demonstrated that cells expressing rat prestin exhibit SCN(-) currents that are proportional to the number of prestin molecules. Variation of the SCN(-) concentration resulted in changes of the current reversal potential that obey the Nernst equation indicating that SCN(-) transport is not stoichiometrically coupled to other anions. Application of external SCN(-) causes large increases of anion currents, but only minor changes in non-linear charge movements suggesting that only a very small percentage of prestin molecules function as SCN(-) transporters under these conditions. Unitary current amplitudes are below the resolution limit of noise analysis and thus much smaller than expected for pore-mediated anion transport. A comparison with a non-mammalian prestin from D. rerio - recently shown to function as Cl(-)/SO(4)(2-) antiporter - and an SLC26 anion channel, human SLC26A7, revealed that SCN(-) transport is conserved in these distinct members of the SLC26 family. We conclude that mammalian prestin is capable of mediating electrogenic anion transport and suggest that SLC26 proteins converting membrane voltage oscillations into conformational changes and those functioning as channels or transporters share certain transport capabilities.

Conserved dimeric subunit stoichiometry of SLC26 multifunctional anion exchangers.

The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits. Co-expression of two mutant prestins with distinct voltage-dependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.