Fig 1.
Assembly state changes of Lil3 by Chlorophyllide and Chlorophyll.
Assembly of the Lil3 protein was investigated in isolated etioplasts (E). Plastids were incubated in the absence (-) or presence (+) of GGPP and upon illumination (L20) or in darkness and presence of Zn-pheide (D20) for 20 min at 25°C. Membranes were solubilized and protein complexes separated by LN-PAGE in a 7.5% acrylamide gel. Native gels were scanned by laser excitation at λ = 633nm (A, Fluorescence). The Cyt b6f band was labeled upon immunological detection of Cyt b6 (A, Cyt b6f). Lil3 protein in fluorescent bands (F1-3) and Lil3 protein in non-fluorescent bands (NF1-4) was identified using polyclonal antibodies directed against Lil3 (B, Lil3 gel-blot).
Fig 2.
Comigration analysis of Lil3, CHS, POR, and Cyt b6 by LN-PAGE.
Plastids were isolated from etiolated barely seedlings illuminated for 10 seconds at 25°C. Membranes were solubilized and protein complexes were separated by LN-PAGE (1D). The fluorescence of bands at 670 nm was analyzed by excitation scanning of gels at 633 nm. The mobility of tetrapyrrol binding proteins Lil3, CHS, POR, and Cyt b6 was investigated by denaturation of the native gel lane and separation of protein subunits using second dimension SDS-PAGE and gel blot analysis (2D). Mobility of fluorescent Cyt b6f bands C1 and C2, and of the Lil3 bands F1, F2 and F3 was labeled according to the antibody signals of the Cyt b6 and the Lil3 subunits. Overlapping mobility of proteins in the 1D gel has been connected by a vertical line. A peptide specific antibody against the C-terminus of Lil3 was employed (S1 File).
Fig 3.
Changes in the protein composition of Lil3 bands.
Protein changes in the molecular mass region of fluorescent native PAGE bands labeled Cyt b6f, F1, F2, and F3 were investigated by comparative MS and bioinformatics analysis (Methods, S1 Table). Gene annotation (gene), protein name (protein) and maximum peptide count number (max) (MS identification) as well as normalized peptide count numbers are listed for each band. The ratio of changes between the dark (D) and Chlide (L, F3), and Chlide plus GGPP (L, F1 and F2) values are plotted (GGPP/Chlide, L/D) for the Cyt b6f (Cyt), the Lil3-F1 (F1) and the Lil3-F2 (F2) band. Gene and band arrangement was mathematically calculated by the position in the Euclidean distance matrix. Cluster numbers (#) were given top down at a minimum similarity cut off (CO) value at 0.766 (dashed line).
Fig 4.
Direct interaction analysis between POR, Lil3, CHS, and GGR.
NMY51cells were co-transformed with BTC-Lil3:1 or BTC-Lil3:2 and, PRN-PORA, PRN-PORB, PRN-PORC PRN-CHS, PRN-GGR (A), with BTC-GGR or BTC-CHS and, PRN-PORA, PRN-PORB, PRN-PORC, PRN-GGR, PRN-CHS, PRN-Lil3:1, PRN-Lil3:2 (B). Serial dilutions of the yeast strain were made to evaluate the specificity of the interaction.
Fig 5.
Determination of Chlide, ChlGG, and ChlPY in fluorescent Lil3 bands.
For identification of Chlide specific fluorescence in protein complexes, isolated etioplasts (1x108) were incubated without (lanes 1, 3) or supplemented with (lanes 2, 4) GGPP on ice for 1 min in darkness, plastids were frozen on dry ice and either maintained in the dark (lanes 1, 2) or exposed to light for 10 seconds (lanes 3, 4) (A). Plastids were lyzed, membranes were solubilized and protein complexes separated by LN-PAGE (3–12% acrylamide gradient) (A). For identification of pigments bound to fluorescent Lil3 bands, pigments were extracted from etioplasts (B) or from gel-bands after separation by 7.5% native PAGE (C). For identification of pigments in etioplast membranes (B), acetone extraction was conducted using etioplasts kept in darkness (B, lane 1), illuminated (B, lane 2), illuminated and incubated with GGPP (B, lane 3), or illuminated in the presence of GGPP and NADPH (B, lane 4), or FPP and NADPH (B, lane 5). Etioplasts isolated after a 10 sec in vivo illumination of etiolated plants (B, lane 6) were extracted and loaded as control. For identification of pigments bound to protein complexes (C), fluorescent Lil3 bands F3 (C, lane 1 and 2), F2 (C, lane 3), and F1 (C, lane 4) were extracted. Pigment synthesis was induced by a 10 s light exposure of etioplasts (C, lane 1) or of etiolated plants (C, lane 2–4). HPTLC and native gels were scanned for fluorescence by laser excitation at λ = 633nm. The position of fluorescent protein complexes Cyt b6f, F3, and of pigments in the gel front (PF) of the native gel (A), and of pigments Pchlide, Chlide, Pchl, ChlGG, ChlPY, ChlF, and unidentified GGPP and FPP dependent tetrapyrrol derivatives (ChlGG* and ChlF*) after HPTLC separations (B, and C) are marked.
Fig 6.
Assembly of proteins POR, Lil3, CHS, and GGR.
The enzymes protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), and geranygeranyl reductase (GGR, EC1.3.1.83) are stromal enzymes (Stroma). Chloropohyll synthase (CHS, EC 2.5.1.62), and the light-harvesting like protein Lil3 are membrane integral proteins of the chloroplast (Membrane). All four proteins are proposed to interact in barley etioplasts during synthesis of phytylated Chl. POR binds protochlorophyllide a (Pchlide) and synthesizes chlorophyllide a (Chlide) in the light. Chlide released from POR (arrow) or chemically supplied, assembles with a protein band containing Lil3 and CHS. At low temperature Chlide and ChlGG are isolated from the F3 band (grey background). At room temperature, fluorescence and molecular mass of Lil3 shifts, GGR accumulates in the bands F1, and F2 (Figs 1 and 3) and F1, F2, and F3 bind ChlPY (Fig 5, grey background). The F2 band accumulates psb29 and D2 indicating that Lil3 participates in the delivery of ChlPY (left arrow). POR, GGR, and Lil3 are detected in all fluorescent complexes F1, F2, and F3 by mass spectrometry or gel-blot analysis (dotted boxes). The presence of CHS, and GGR in fluorescent bands is indicated by accumulation of ChlGG and ChlPY. Lil3 is shown to directly interact with CHS, and POR in a split ubiquitin based Y2H screen (Fig 4). No direct interaction between GGR and Lil3 or POR and CHS was identified.