Fig 1.
Summary of experimental set-up.
(A) Membrane proteins and mRNA were isolated from Chara australis internodal cells incubated at ambient day/night cycles with 14 h light (contr). mRNA was subjected to RNAseq analysis and the assembled unigenes (nt, nucleotide sequence) were annotated to functional classes with BLAST using the databases GO, COG, KEGG and NR. Additionally, Mercator software classified the unigenes into functional categories (BIN classes), by combining BLAST searches and InterProScan using the databases TAIR, Uni/SwissProt, COG, cdd and InterProScan. Unigenes were also translated into amino acid sequences (aa), thus serving as a protein database in addition to TAIR for the membrane proteome analysis with MaxQuant software. See Material & Methods section for abbreviations of databases. (B) Acidic (acid) and alkaline (alk) areas of single internodal Chara cells were separated and collected for membrane preparation. Alkaline areas were visualized with phenol red. To facilitate collection, internodal cells were fixed by the weight of stainless steel nuts. Bar = 5 mm.
Fig 2.
Total membrane fractions of Chara internodal cells.
(A) Proteins of membrane fractions (MF) were separated by SDS-PAGE (10%), stained with Coomassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H+ ATPase. 30 μg protein per lane. Numbers on the left refer to molecular weight markers in kDa. Only the upper part of the PVDF membrane was used, the lower part was probed for immunodetection of low molecular weight proteins. (B) Immunodetection of selected organelle marker proteins for vacuoles (VHA-ɛ, H+ PPase), ER (BiP2), plasma membrane and endosomal compartments (ARA6) or cytosol (tubulin, GRF 14-3-3). Proteins of the MFs were separated by preparative SDS-PAGE (12.75% or 10% for GRF and ARA6), plotted onto PVDF membranes cut into 3 mm strips and detected with the respective antibodies. 10 μg protein per strip. Molecular weight markers are given in kDa. Arrow heads indicate the expected position of the respective protein.
Fig 3.
ATP hydrolysis activity in Chara MF.
The specific ATP (3 mM Mg-ATP) hydrolysis activity was determined by detecting the released phosphate in the presence of 1 mM azide and 100 nM bafilomycin A to inhibit F- and V-type ATPases. Vanadate and fusicoccin were added to inhibit and to stimulate P-type ATPases respectively. Mean ± S.D. of 3–6 experiments. Probabilities (p) were determined with Student’s t-test.
Fig 4.
Topology of the Chara PM H+ ATPase.
The sequence of unigene CL2034.contig1 was translated into amino acid sequence. Transmembrane domains were predicted by TMPred ([50]; (www.EXPASY.org)) and drawn with PROTTER (wlab.ethz.ch/protter). Peptides identified by mass spectrometry analysis, are given as orange diamonds.
Fig 5.
Differences in charasome abundance and protein expression in alkaline and acidic regions of Chara cells.
(A) Left image pair: FM1-43-labelled charasomes (green fluorescence) and chloroplasts (bright field image) at an acidic band. Right image pair: FM1-43-stained charasomes are absent from the alkaline band; the bright field images show the chloroplasts. An FM1-43-stained internodal cell was cut into acid and alkaline regions as described in Materials and Methods guided by pH banding pattern visualized by phenol red. Cell fragments were mounted in artificial fresh water and examined in the CLSM. Bar = 20 μm (B) Proteins of membrane fractions (MF) and cytosolic fractions (CF) obtained from acidic (ac) and alkaline (alk) regions were separated by SDS-PAGE (10%) and stained with Comassie Brilliant Blue (CBB) or blotted onto PVDF membranes for immunodetection of the plasma membrane H+ ATPase using only the upper part of the membrane. 15 μg protein was loaded per lane. Numbers on the left refer to molecular weight markers in kDa. (C) Immunodetection of selected higher and lower molecular weight proteins from the membrane fraction (MF) as indicated. 10 μg protein was loaded per lane, 10% gel. PVDF membrane was cut into upper and lower halves at 50 kDa. Molecular weight markers given in kDa.
Fig 6.
Differential proteome of the alkaline and acidic regions of Chara cells.
Peptides were searched against the custom-made unigene Chara database (see also S1 Table) and assorted into the appropriated BIN classes. The fraction of total peptides (fot) for the respective BIN class was calculated. Ratios were subtracted by 1 to indicate enriched classes and depleted classes in the acidic regions by positive and negative values, respectively. CHO = carbohydrate metabolism, TCA = tricarboxylic acid cycle, PS = photosynthesis.
Table 1.
List of identified proteins of the BIN class ‘34 transport’ by LC-MS/MS.
Table 2.
List of identified proteins belonging to the BIN class ‘31.4 cell.vesicle transport’.
Fig 7.
Distribution of individual peptides of the alkaline and acidic regions of Chara cells.
Peptides were annotated using TAIR database and categorized into BIN classes as indicated. Differential expression was analysed with cRACKER software. Dotted line shows threshold of p = 0.05. Arrow indicates the PM H+ ATPase peptide.
Fig 8.
Distribution of H+ transporters between acidic and alkaline regions.
H+ transporting proteins predicted for plasma membrane localisation (Table 1) were drawn in the scheme according to their abundance as dark red or bright red symbols representing high and low abundance, respectively. The cell wall (CW) is sectioned in acidic (H+ in black) or alkaline (H+ in grey) regions with the plasma membrane (PM) underneath and charasomes drawn as invaginations in the acidic region. The ion transporters are labelled as following: 1 = APC (amino acid/polyamine/organo-cation) transporter, urea/H+ symporter DUR3 (at5g45380) 2 = ammonium transporter (NH4+ or NH3 plus H+) AMT1;2 and AMT1;5 (at1g64780, at3g24290)3 = sucrose/H+ symporter SUC3/SUT2 (at2g02860) 4 = sulphate/H+ symporter SULTR1 (at1g78000) 5 = Na+/cation transporter HKT1 (at4g10310) 6 = cation/H+ exchanger CHX18/19 (at5g41610, at3g17630) 7 = P-type H+ ATPase, PM H+ ATPase (at5g57350).