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.