Inversion of plant plasma membrane vesicles for the recovery of phosphopeptides from the cytoplasmic face.A, low concentrations of the detergent Brij-58.

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Inversion of plant plasma membrane vesicles for the recovery of phosphopeptides from the cytoplasmic face.A, low concentrations of the detergent Brij-58 generate sealed inside-out vesicles, as visualized by ATP-dependent proton pumping activity in Brij-58-pretreated plasma membrane vesicles. Inversion of plant plasma membrane vesicles for the recovery of phosphopeptides from the cytoplasmic face.A, low concentrations of the detergent Brij-58 generate sealed inside-out vesicles, as visualized by ATP-dependent proton pumping activity in Brij-58-pretreated plasma membrane vesicles. The decrease in absorption of acridine orange reflects the acidification of the vesicle lumen. Addition of 1 μm nigericin (Nig) abolishes the established pH gradient. B, two-dimensional TLC/TLE of phosphopeptides from in vivo-labeled plasma membranes that have been inverted with Brij-58 and “shaved” with trypsin. The origin (site of sample application) is indicated by “+.” Signals that are strongly induced in elicited versus control samples are marked with upward pointing arrowheads, unchanged signals (showing even loading) with arrowheads pointing right. Thomas S. Nühse et al. Mol Cell Proteomics 2003;2:1234-1243 © 2003 The American Society for Biochemistry and Molecular Biology