Automating Single Subunit Counting of Membrane Proteins in Mammalian Cells

Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope. Background: Although powerful, single subunit counting is time-consuming, prone to user bias, and largely restricted to Xenopus expression. Results: PIF is an automated analysis program that identifies subunit stoichiometry of any fluorescently tagged membrane protein from TIRF recordings. Conclusion: PIF is accurate to more than 90% even in noisy data typical for mammalian expression system. Significance: The PIF approach is generalizable to any membrane protein and TIRF microscope.