Quaternary structure of the yeast pheromone receptor Ste2 in living cells

Abstract

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10 to 3.5·10molecules/nm), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10 to 1.4·10molecules/nm). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

FRET from single to multiplexed signaling events

“…The biologically relevant metric in these cases is the quantity of complexes of a defined composition in a cell. Using spectral unmixing, FRET coupling in mixed donor–acceptor complexes was resolved (Raicu 2007; Singh et al 2013; Stoneman et al 2016). …”

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

“…Since the laser wavelength of 860 nm (selected as optimal for donor excitation) also caused slight excitation of the acceptor, we did not attempt to compute the FRET efficiency for every image pixel from the donor and acceptor fluorescence maps (Raicu and Singh, 2013), or else the FRET efficiency map would have been contaminated by such spurious excitation. Instead, we used a method introduced recently, which employed two excitation wavelengths (860 and 960 nm) to account for the direct excitation of the acceptor and compute the average FRET efficiency for regions of interest (at the plasma membrane; Stoneman et al, 2016). In order to reduce noise and avoid singularities at pixels with zero intensity values, a signal-to-noise threshold of 1.5 SD was applied to FRET efficiency calculations.…”

A general method to quantify ligand-driven oligomerization using single- or two-photon excitation microscopy

“…Imaging using two-photon microscopy. Fluorescence images (800×480 pixels 2 ) were acquired using a two-photon optical micro-spectroscope 31,40 comprised of a Zeiss Axio Observer inverted microscope stand and an OptiMiS detection head from Aurora Spectral Technologies. A mode-locked laser (MaiTai TM , Spectra Physics), which generated 100 fs pulses, was used for fluorescence excitation at 960 nm.…”