1. Volume 2, Issue 6, Pages 1410-1424 (November 2009)
Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex, the CF1CF0–ATP Synthase 
Benz Monique , Bals Thomas , Gügel Irene Luise , Piotrowski Markus , Kuhn Andreas , Schünemann Danja , Soll Jürgen , Ankele Elisabeth  
Molecular Plant 
Volume 2, Issue 6, Pages (November 2009)
DOI: /mp/ssp095
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Characterization of Arabidopsis alb4 Loss-of-Function Mutants.
Alb4 levels were measured in two independent Arabidopsis alb4 TILLING mutant lines. As a control, a T-DNA insertion line (#199) expressing reduced levels of Alb4 as described (Gerdes et al., 2006) was supplemented.
(A) Schematic representation of the Arabidopsis alb4 gene. Exons are represented as solid boxes and introns as thin lines. Positions of point mutations in the alb4 gene are indicated by open triangles. In line #437, translation terminates after the third exon, owing to a premature stop codon, whereas line #771 shows aberrant splicing behavior due to a G→A transition in intron 2.
(B) Expression of alb4 in wild-type ER, line #437 and line #771. Due to alternative splicing, two fragments are amplified by alb4-specific primers. The larger sequence codes for full-length alb4 including intron 2 whereas the second sequence lacks most of the exon 2 sequence.
(C) Chloroplasts (corresponding to 50 μg of total protein) were prepared from mature leaves of 3-week-old wild-type and alb4 mutant plants, separated by SDS–PAGE, and subjected to immunoblot analysis using an antibody specific for Alb4 (upper panel). A specific antiserum against Alb3 was used as a loading control (lower panel).
(D) Growth phenotypes of 10-day-old and 3-week-old alb4 mutants. The wild-type lines wild-type ER and Col0 as well as the three alb4 mutant plants (#199, #437, #771) were germinated and grown on soil under long-day (16 h light/8 h dark) conditions in a climate chamber for 10 d and 3 weeks.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
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3. Figure 2 Complementation of a Conditional E. coli yidC Mutant by Alb4.
The conditional E. coli yidC mutant strain MK6, which normally requires arabinose for growth, was transformed with one of four different plasmids: the empty vector pGZ (lane 1), pGZ–YidC carrying the yidC gene under the control of an IPTG-inducible promoter (lane 2), pGZ–Alb4ΔC encoding a truncated alb4 cDNA that lacks the coding sequence for the C-terminal region of Alb4 (lane 3), and pGZ–Alb4 encoding the full-length alb4 cDNA (lane 4). Transformed cells were grown under control conditions on LB plates containing (A) arabinose (Ara) or (B) arabinose supplemented with 1 mM IPTG, and under selective conditions on LB plates containing (C) glucose (Glc) or (D) glucose supplemented with 1 mM IPTG. Cell density is indicated on the right (10−1–10−5).
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
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4. Figure 3
Immunoblot Analysis of Steady-State Levels of ATPase Subunits.
Chloroplasts were isolated from wild-type and alb4 mutant plants grown for 3 weeks on soil. Chloroplast samples equivalent to 10 μg of protein were separated by SDS–PAGE (15% acrylamide, 4 M urea), immunoblotted, and analyzed with specific antisera raised against (A) CF0II, CF0III, CF1γ, CF1δ, CF1ϵ, CF1α, CF1β, and Tic62 as a loading control.
(B) Chloroplast samples of the T-DNA insertion line treated as above were analyzed using antisera against CF0III and CF1δ.
(C) A Coomassie-stained SDS–PAGE is presented to show equal loading of TILLING samples.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
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5. Figure 4
Immunodetection of Sub-Complexes of the ATP Synthase Resolved by 2D BN-/SDS–PAGE.
Chloroplasts from 3-week-old wild-type and alb4 mutant plants (equivalent to 15 μg of chlorophyll) were solubilized with 1% dodecylmaltoside and first separated on 5–12% (w/v) BN–PAGE gels. Gel slices containing selected protein complexes were then subjected to SDS–PAGE (15% polyacrylamide, 4 M urea), followed by immunoblot analysis using antisera raised against ATP synthase subunits specific for the hydrophilic CF1 sub-complex CF1α/b (A) and CF1γ (B), or the CF0III subunit (C) of the membrane-embedded CF0. Differences in ATP synthase complexes between wild-type (upper panel) and alb4 mutants (437, 771) are indicated by arrows and circles, respectively. α, β indicate CF1α and CF1β subunits. The asterisk (*) indicates an unspecific signal obtained with the CF1γ antiserum that was used as an internal loading control. Membranes from one experiment (wild-type and mutants) were exposed onto one X-ray film to assure equal exposure.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
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6. Figure 5
Photophosphorylation Takes Place at Reduced Levels in alb4 Mutant Lines.
(A) Chloroplasts (equivalent to 5 μg of protein) isolated from leaves of 3-week-old plants were lysed and incubated with 32P and ADP. The reaction was stopped with EDTA, centrifuged, and aliquots of the supernatant applied onto a thin-layer chromatography plate. Radioactivity was monitored by X-ray films.
(B) Radioactive spots were isolated and 32P activity counted in a scintillation counter. Data of four to six biological replica were used for calculation. Data obtained for wild-type ER and wild-type Col0 were set to 100%. Average percentage of activity is given for each mutant line compared to its corresponding wild-type.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Alb4 Co-migrates with the ATP Synthase Complex but Not With Alb3.
(A) Thylakoid membranes (equivalent to 10 μg of chlorophyll) isolated from leaves of 3-week-old wild-type plants were solubilized with dodecylmaltoside, separated by 2D BN–/SDS–PAGE (5–12% polyacrylamide, 4 M urea), blotted onto filters, and analyzed with antisera specific for Alb4 and the ATP synthase subunits CF0III, CF1α/β, and CF1γ as indicated at the left. The asterisk (*) indicates an unspecific signal obtained with the CF1γ antiserum. Marker sizes are given in kDa at the top.
(B) Thylakoid membranes were solubilized with dodecylmaltoside and fractionated by gel filtration on a Superose 6 10/300 GL column. Aliquots of the fractions, together with solubilized thylakoid membranes, were subjected to immunoblot analysis with the antibodies indicated at the left. Marker sizes are given in kDa at the top. TM, solubilized thylakoid membranes.
(C) Thylakoid membranes were subjected to immunoprecipitation analysis using specific antibodies against Alb4, Alb3, and cpSecY. Aliquots of the immunoprecipitates (IP) and solubilized thylakoid membranes (TM) were separated on SDS–PAGE gels and analyzed by immunoblotting with antisera as indicated.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
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8. Figure 7 Alb4 Interacts Physically with Subunits of the ATP Synthase.
(A) Thylakoid membranes were incubated with (+) or without (–) the chemical cross-linker sMBS for 30 min at 25°C. After the cross-link reaction, proteins were separated by SDS–PAGE, followed by immunoblot analysis with antibodies specific for Alb4, Alb3, and CF1β. The specific 170-kDa cross-linked product is indicated by an asterisk (*). Marker sizes are given in kDa on the left.
(B) Arabidopsis mesophyll protoplasts were transiently transfected with constructs coding for Alb4 and CF0II fused to the C- and N-terminal fragments of the yellow fluorescent protein (YFP), respectively. In the control reactions, protoplasts were transfected with a combination of the constructs and the corresponding control plasmid pSpyne-TS(rbcs) or pSpyce-TS(rbcs). The control plasmids pSpyne-TS(rbcs) or pSpyce-TS(rbcs) code for the transit sequence of the small subunit of Rubisco, resulting in the import of the nYFP or cYFP into the chloroplast. Confocal images are shown for YFP fluorescence, chlorophyll autofluorescence, and an overlay of both.
(C) Protoplasts transfected with Alb4–cYFP and CF0II–nYFP were lysed, centrifuged, and the supernatant (sup) precipitated with TCA. The membrane pellet was washed, centrifuged, and the supernatant (wash) was precipitated with TCA. Samples were separated on SDS–PAGE and subjected to immunoblot analysis. For the controls, untransfected protoplasts were used.
Molecular Plant 2009 2, DOI: ( /mp/ssp095)
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions