Non-invasive tools for analysis of plasma membrane protein topology in living cells.

Membrane protein topology studies offer guidance to membrane protein structure, folding, and function, serving as a credible scaffold for designing site-directed mutagenesis and biochemical experiments, helping to identify functionally significant extracellular and intracellular regions, modeling three-dimensional structures, and building reliable mechanistic models. Membrane protein structure as a function of given lipid composition and physiological state of the cell is best probed in whole intact cells. A described simple and advanced immunofluorescence protocol applied to the transmembrane orientation of extramembrane domains permits a topology analysis of plasma membrane proteins in their native state in living unperturbed eucaryotic cells. The accessibility of native epitopes to corresponding antibodies is determined in intact and permeabilized cells to establish their extra- or intracellular or localization respectively. The ability of the given antibody to bind the epitope in intact live and permeabilized cells is then assessed routinely by intact and permeabilized cell immunofluorescent confocal microscopy or fluorescence flow cytometry parametric analyses in several hours. To ensure that the observed immunofluorescence is entirely a result of the binding of antibodies, cells are alive and the plasma membrane is intact, plasma membrane integrity is routinely monitored by co-incubating the cells with a cell membrane-impermeable probe, propidium iodide. Accordingly, plasma membrane side-specific immunostaining analysis was restricted to the propidium iodide-negative, non-permeabilized cell population. The strength of this technique is its simplicity since each native epitope is unique and there is no need to mutate any endogenous sites, introduce new epitopes, or engineer single, dual, or split colorimetric enzymatic reporters. Aside from its simplicity, the advantage of this approach is that the topology is documented in the context of full-length and fully biologically active membrane protein molecules, and topology mapping is carried out using whole live cells, thereby avoiding problems related to cell fixation or the conversion of cells into membrane vesicles with a uniform orientation. The protocol can be universally adapted to any cellular system to systematically map a uniform topology of target membrane protein.