Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: Co-purification of KOC1 with TOC159 and TOC components. Sequential fractions of NTAP-TOC159 IgG-affinity purification were analyzed by Western blotting. The membrane was probed with antibodies against KOC1, CBP, TOC75, TOC33, TIC110, LHCB2, and PGL35 (L, load; Ft, flow-through;W1, first wash; W5, last wash; TEV, eluate). 50 μg of proteins (load, flow-through, and first wash) or 10% of the fractions (last was and eluate) were loaded. Several identical blots were used for immunoblotting. TAP:WT was used as a negative control.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Copurification, Affinity Purification, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: Co-purification of TOC components with KOC1. A, sequential fractions of NTAP-KOC1 IgG-affinity purification were analyzed by Western blotting. The membrane was probed with antibodies against KOC1, TOC159, TOC75, TOC33, TIC56, and LHCB2 (T, total; L, load; Ft, flow-through; W1, first wash; W5, last wash; TEV, eluate). 50 μg of proteins (total, load, flow-through, and first wash) or 10% of the fractions (last wash and eluate) were loaded. Several identical blots were used for immunoblotting. TAP:WT was used as a negative control. B, interaction networks of KOC1 using the list of co-immunopurified proteins and the STRING 10.0 program.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Copurification, Affinity Purification, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: KOC1 sequence analysis and mutant characterization. A and B, the amino acid sequence of KOC1 contains a kinase domain (orange; amino acids (aa) 39–306). Underlined amino acids 45–53 are conserved in the catalytic domain; amino acid 74 is predicted to bind ATP. In the HERC2-related region (blue; amino acids 376–531), underlined amino acids share homology with the KEG protein. A predicted transmembrane stretch (TM; amino acids 549–572) is highlighted in red. C, images of 2-week-old WT, koc1-1, koc1-2, and NTAP-KOC1:koc1-1 plants grown in vitro. D, total protein extracts of 2-week-old plants (WT, koc1-1, koc1-2, and NTAP-KOC1) were separated by SDS-PAGE and transferred to nitrocellulose membrane. Antibodies against TOC159A, TOC75, TOC120, KOC1, TOC33, and TIC110 were used. Actin was used as a loading control. *, bands corresponding to overexpressed NTAP-KOC1.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Sequencing, Mutagenesis, In Vitro, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: Localization of KOC1 at the outer membrane of the chloroplast. A, confocal microscopy images of an isolated representative protoplast expressing YFP-KOC1. Chlorophyll fluorescence in red identifies chloroplasts, the signal of YFP-KOC1 appears yellow, and intact protoplasts were visualized by bright field. The merge shows the overlay of chlorophyll, YFP-KOC1, and bright field images. Scale bars, 10 μm. B, total membranes from NTAP-KOC1 chloroplasts were separated on a continuous sucrose gradient (5–45%), and 37 fractions were collected. Proteins from uneven fractions were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against CBP (1), TOC159A (2), TOC75 (3), LHCB2 (4), and PGL35 (5). C, Col-0 chloroplasts were subjected (+) to thermolysin treatment or not (−), TX1000 solubilization, and alkaline extraction (Na2CO3) (P, pellet; S, supernatant). Samples were separated by SDS-PAGE, transferred to nitrocellulose, stained with Amido Black (lower part), and probed with antibodies against KOC1, TOC75, and TIC40. LSU, large subunit.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Confocal Microscopy, Isolation, Expressing, Fluorescence, SDS Page, Staining

Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: Phosphorylation of the A-domain by KOC1 and its presence in TOC159. A, TOC159A was incubated with KOC1 isolated from NTAP-KOC1:koc1-1 plants (lane 1) or the same but heat-denatured KOC1 (De-KOC1) (lane 4) in the presence of [γ-33P]ATP. B, purified TOC159A was incubated with [γ-33P]ATP and with recombinant full-length KOC1 purified from bacteria (lane 3). C, purified TOC120A (lane 2) and TOC132A (lane 3) were incubated with [γ-33P]ATP and KOC1 isolated from NTAP-KOC1:koc1-1 plants. In A–C, the samples were separated by SDS-PAGE and analyzed using a phosphorimaging system (Molecular Imager FX) and Quantity One 4.6 software. KOC1, TOC159A, TOC120A, and TOC132A were incubated alone with [γ-33P]ATP as negative controls. D, total protein extracts of seedlings ppi2 (lane 1), WT (lane 2), NTAP-TOC159:ppi2 (lane 3), and NTAP-TOC159-cmyc:ppi2 (lane 4) were analyzed by Western blotting. The membrane was consecutively probed with antibodies against c-myc and rabbit IgG. The data shown are representative experiments of several replicates.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Incubation, Isolation, Purification, Recombinant, SDS Page, Software, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

doi: 10.1074/jbc.M117.776468

Figure Lengend Snippet: Requirement of KOC1 for efficient chloroplast protein import and de-etiolation. A and B, isolated chloroplasts from koc1-1 and Col-0 (WT) plants were incubated with [35S]Met-labeled preproteins of pSSu in A and E1-α in B. The preproteins were incubated with chloroplasts, and import was allowed to proceed for 0, 5, and 15 min (0′, 5′, and 15′); one sample was treated with thermolysin after 15 min (15′T). Proteins from chloroplasts were separated by SDS-PAGE followed by phosphorimaging analysis. The graphs show the quantification of the bands corresponding to imported mature SSu and E1α at 0, 5, and 15 min averaged over three technical replicates. The amount of mature protein imported into WT chloroplasts at 15 min was arbitrarily set to 100%. The qualitatively similar results were obtained in five independent experiments. C, images of surviving koc1-2, koc1-1, WT, and NTAP-KOC1:koc1-1 plants upon exposure to long-day conditions for 2 weeks after etiolation for 6 days in dark. The germination and survival rates were calculated. The germination rate was around 100% for all genotypes. The survival rates were as follows: WT, 34.6%; NTAP-KOC1:koc1-1, 22.5%; koc1-2, 8.8%; and koc1-1, 12.5% (Student's t test: **, p value <0.01; *, p value <0.05 (n = 80 for koc1-1 and koc1-2; n = 240 for WT)). Error bars represent S.D. This experiment was repeated three times with comparable results. rel. amt., relative amount.

Article Snippet: For anti-KOC1 antibody production, two forms of recombinant KOC1 (KOC1(1–343) and KOC1(1–547)) purified by Ni 2+ -NTA-affinity chromatography were pooled and injected into rabbits for polyclonal antibody production (Eurogentec, Seraing, Belgium).

Techniques: Isolation, Incubation, Labeling, SDS Page