Enrichment of plasma membrane
For the PM enrichment of potato roots, all six vessels obtained from each independent biological experiment were pooled. The two phase PM enrichment protocol described by Hynek, Svensson, Jensen, Barkholt, and Finnie (2006) was modified to treat potato roots as described recently (Jozefowicz, Matros, Witzel, & Mock, 2018). Briefly, the microsomal fraction of potato roots was obtained by differential centrifugation (15 min 10,000x g , 50 min 50,000 x g , 4 °C). Subsequently, 3 g of membrane suspension were loaded onto 9 g two-phase mixture composed of 6.2% Dextran T500, 6.2% PEG 3350, 8 mM KCl pH 7.8, 300 mM sucrose, 5 mM potassium phosphate buffer. Loosely bound and soluble proteins trapped in vesicles were removed by washing the PM enriched upper phase pellet in Brij-58 buffer (0.33 M sucrose, 0.2 M KCl, 5 mM phosphate buffer (pH 7.8), 0.2% (w/v) Brij-58) (Alexandersson, Saalbach, Larsson, & Kjellbom, 2004). Protein quantification was performed according to Bradford (1976). Proteins (5 µg per fraction) were separated by electrophoresis through 11.25% sodium dodecyl sulphate polyacrylamide gels (Laemmli, 1970) and subsequent Western blot analyses performed as described elsewhere (Amme et al., 2005). The following antisera were used: anti-H+-ATPase (diluted 1:3000), anti-V-ATPase (1:3000), anti-VDAC (voltage dependent anion channel, 1:2000), anti-BiP (luminal binding protein, 1:8000) (Agrisera, Vannas, Sweden) and self-raised anti-MDAR (monodehydroascorbate reductase, 1:1000). The universal secondary antibody used was the goat polyclonal anti-rabbit conjugated with IRDye 800CW (1:15000), which enabled infrared fluorescence detection via a LI-COR Biosciences scanner (Bad Homburg, Germany). Relative quantification of the fluorescence signal was performed using Odyssey 3.0 software and mean values were compared using the Student’s t -test.