Mesembryanthemum crystallinum and Arabidopsis thaliana were grown in hydroponics and soil culture, respectively, as described in [10, 44]. Growth conditions were 120 μmol quanta m-2 s-1, 60% relative humidity, 20°C, and a daily photoperiod of 12 h duration. Rosette leaves from 3- to 5-week-old Arabidopsis plants were taken for protoplast transfection. Zea mays and Hordeum vulgare were germinated on filter paper in the dark at 25°C for 48 h. Cells were isolated from the first 2 mm of the growing root tip. Onion epidermis was stripped from onion bulbs obtained from a local market.
Leaves (50 g) of M. crystallinum were homogenized in a buffer containing 250 mM sucrose, 50 mM Tris-Cl, pH 8.0, 4 mM ethylenediamine tetraacetic acid (EDTA), 4 mM dithiothreitol and a few crystals of phenylmethylsulfonylfluoride . As indicated in a set of experiments, either NaCl was added at 100 or 500 mM concentration or complete protease inhibitor® cocktail (Roche, Mannheim, Germany) was added throughout the procedure. Following differential sedimentation and gradient centrifugation, tonoplast enriched membranes were recovered from a 30%/35% sucrose interphase, sedimented, frozen in liquid nitrogen and stored at -80°C.
Gel electrophoresis and Western blot detection
Membrane proteins were separated on 12.5% sodium dodecylsulfate polyacrylamide gels, transferred to nitrocellulose and probed with anti-VHA-E , anti-VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany) raised in rabbit or anti-VHA-a raised in guinea pig. Following incubation with primary and secondary antibody conjugated with peroxidase, detection was achieved with the lumilight® system according to the supplier (Roche, Mannheim, Germany).
For immunoprecipitation, membranes were solubilised in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% (v/v) Triton X-100, 5 μl anti VHA-a antiserum was added, and the samples were shaken at room temperature for 45 min. Then 150 μl protein A-sepharose equilibrated in the same buffer was added. After 15 min, the suspension was placed on a cushion of 1 ml of 40% sucrose and spun at 10,000 × g for 1 min. The sediment was washed thrice with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1 % (w/v) SDS, and finally once in 125 mM Tris-Cl, pH 6.8. The sediment was boiled in loading buffer and analysed by Western blot using rabbit antisera raised against VHA-E or A.
Anti-VHA- a antibody preparation and other antibodies used in this study
Two antibodies against specific domains of VHA-a were raised in rabbits, and denominated anti-VHA-aN-term and anti-VHA-aMemb. For both the corresponding cDNA fragments of Mc-VHA-a were amplified by PCR using primer combinations a-nterm-f (ATG CGA TCG GAG CCG ATG CAA) and a-nterm-r (TTC ACC CAA CTC ATC GGT GG) encoding the 42 N-terminally located fragment, and a-memb-f (CTT CCA AAG CCC TTT ATT ATG) and a-memb-r (TCA CTC ATG TCC ACC ATG TCA ATC) encoding the polypeptide loop of about 13 kDa located between transmembrane domain 3 and 4 according to the topological model of Vph1p of S. cerevisiae . The gene fragments were cloned into the vector pCR-T7-NT-Topo (Invitrogen, The Netherlands) and transformed into E. coli JM109. The 6x-his-tagged proteins were expressed, purified by chromatography on Ni-nitrilotriacetate columns, separated by preparative SDS-PAGE, excised as protein bands, eluted and used for immunization (Pineda, Berlin). In addition, antisera against subunits VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany), VHA-E, calreticulin (, kindly provided by Andrew Smith, Oxford, UK), Jim 84 ( kindly provided by Chris Hawes, Oxford, UK), γ-TIP (; kindly provided by John C Rogers, Washington State University, USA) were used in the co-localization studies.
Construction of fusions between VHA subunits and variants of green fluorescence protein (GFP)
Mc-VHA-a and -c were cloned into the vectors pECFP/pEYFP (Clontech, Palo Alto, USA) in a site-directed manner after amplification from cDNA  using the primers a-ges-BamHI-f (AAA AGG ATC CAT GCG ATC GGA GCC GAT GCA A) and a-ges-NcoI-r (AAA AAC ATG GCC TCT TCT TCT TCA CCA ATC GT), McVHA-c with c-ges-BamHI-f (AAA AGG ATC CAT GTC AAC CGT CTT CAA TGG) and c-ges-NcoI-r (AAA ACC ATG GCT GCC CTT GAC TGT CCA GCT CG). Mc-VHA-A and Mc-VHA-H were cloned as described in . The constructs were introduced into the vector p35SGFP , so that the chimeric genes were placed under control of the 35S promoter and the original GFP gene was lost. The same strategy was used to produce Mc-VHA-A, -B and -H gene fusions with variants of GFP.
Protoplast isolation and transformation methods
Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCI2, 4 mM morpholinoethane sulfonic acid, KOH , phl 5.7) and checked for sufficient intactness in the microscope. In short, 1 mm leaf slices of 3- to 5-week-old Arabidopsis plants were vacuum-infiltrated and cell walls were digested in media containing 1.5 % (w/v) cellulase R10 and 0.4 % (w/v) macerozyme R10. Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCl2, 4 mM morpholinoethane sulfonic acid, KOH, pH 5.7) and checked for sufficient intactness in the microscope. 110 μl PEG-medium (4 % (w/v) polyethylene glycol 4000, 0.2 M mannitol, 0.1 M CaCl2) and 20 μl plasmid DNA (3 μg/μl) were added to 100 μl protoplast suspension. The samples were incubated at room temperature for 15 min and then consecutively diluted with 0.5, 1, 2 and 4 ml W5-medium with 15 min incubation steps in between (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM morpholinoethane sulfonic acid, KOH, pH 5.7). Following 24 h incubation at 25°C, sedimented protoplasts were used for analysis.
Cells of onion epidermis were placed on filter paper soaked with one-strength MS basal medium in petri dishes and were transiently transformed with a biolistic approach. Gold particles (1.6 μm, 60 mg/ml) were suspended in 50 % glycerol. 8.33 μl of the suspension were mixed with 8.33 μl plasmid DNA (1 μg/ μl), 8.33 μl 2.5 M CaCl2, 3.33 μl 0.1 M spermidine. Sedimented gold particles were consecutively washed with 70 % and 100 % ethanol and resuspended in 8 μl 100 % ethanol, loaded on a macro carrier for transformation with the Particle Delivery System using a rupture disc of 1100 psi (PDS-1000/He, Biorad, Hercules, USA). The distance between macrocarrier and tissue was 12 cm. The epidermis tissue was incubated for about 20 h at room temperature in the dark prior to analysis.
Immuno-fluorescence labelling and image acquisition by confocal laser scanning microscopy (CLSM)
Immuno-labelling was performed according to . In brief, cells were fixed in 3.7 % para-formaldehyde (10 mM MgSO4, 10 mM EGTA, 1 × phosphate buffered saline, pH 6.8), washed, permeabilised in 0.5% Triton X-100 and washed again. Following blocking of non-specific binding sites with 1 % bovine serum albumin, primary antibody was added for over night at 4°C. Washed samples were incubated with secondary antibody labelled with Cy3, Cy5 or FITC for 1 h. Double labelling was performed by combined application of primary antibodies from rabbit and guinea pig. Slides were mounted with Citifluor Mounting Medium. Fluorescence analysis was performed with a confocal laser scanning microscope Leica TCS-SP2 (Leica, Heidelberg, Germany) equipped with three lasers and excitation wavelengths of 458, 476, 488, 514, 568 and 633 nm. The double dichroic mirror DD488/543 was used for fluorescein isothiocyanate (FITC), and for Cy5 the triple dichroic mirror TD488/543/633 was used. Background was controlled and photomultiplier voltage (800 V) selected for maximum sensitivity in the linear range.
Immunogold-labelling and electron microscopy
Cells were fixed in 2.5% glutaraldehyde in EM buffer (50 mM KH2PO4, 50 mM NaH2PO4, pH 7.0) for 45 min, washed with EM buffer and dehydrated with a series of increasing concentration of acetone. Samples were embedded in epoxyresin (Transmit EM, TAAB laboratories equipment, Berkshire, Great Britain), cut into ultra-thin cross-sections of 60–70 nm and immobilized on 200 mesh gold nets. Immuno-decoration was performed with antibody diluted in Tris-buffered saline (TBS, 10 mM bovine serum albumin and 0.05 % (w/v) NaN3) for an hour. Samples were washed five times and incubated with secondary antibody conjugated to 15 nm gold particles. The samples were stained with 0.1 % (w/v) uranyl acetate for 5 s and afterwards with 2 % lead citrate. The samples were analysed with an electron microscope (H500, Hitachi, Japan) at 75 kV.
Confocal microscopy of GFP-fusion proteins and FRET-measurement
Transformed protoplasts and onion epidermis cells were examined for the localisation of the CFP/YFP-fused proteins using the same CLSM set-up as mentioned above. Autofluorescence of 10 protoplasts, as well as reference spectra of YFP and CFP-derived fluorescence were recorded in the spectral range of 480 to 700 nm, averaged and used for corrections. Excitation was recorded at 458 nm (CFP and FRET) and 514 nm (YFP), respectively. Scan speed was 800 Hz. Acceptor dye was bleached with 100 % laser intensity. Emission spectra were recorded and averaged from 20 transformed protoplasts. For a first estimate of transfer efficiency, a Foerster radius for green fluorescence protein variants of Ro = 5 nm  was used to calculate the donor/acceptor distance via the equations E = (ICFP/bleached - ICFP/unbleached)/ICFP/unbleached and R = ((Ro6/E)-Ro6)1/6, where E is the transfer efficiency, and ICFP the fluorescence emission intensity in the CFP peak.