Localization of the AtYLMG1-1 protein. (A) Immunoblot analysis showing the chloroplast localization of AtYLMG1-1. Total proteins extracted from whole plants and isolated chloroplasts (cp) from the wild type were analyzed with the anti-AtYLMG1-1 antibodies. Fifty micrograms of protein were loaded in each lane. The Rubisco small subunit (Rubisco SSU) was detected by CBB staining as the quantitative control. (B) Localization of AtYLMG1-1 in the chloroplast. Chloroplasts were lysed in hypotonic solution and separated into pellet and supernatant fractions by centrifugation. The total chloroplast protein (total cp), pellet (pellet), and supernatant (sup) fractions were analyzed. TOC34 was detected as a marker of the membrane protein and the Rubisco small subunit was detected as a marker of the stromal protein. (C) Localization of AtYLMG1-1 in the chloroplast membranes. Isolated chloroplasts from the wild type were lysed and separated into thylakoid and envelope membranes. Proteins of the total chloroplast (total cp), the envelope fraction (env), and the thylakoid fraction (thy) were examined with the anti-AtYLMG1-1 antibodies. Lhcb1 was detected as a marker of the thylakoid protein and TOC34 was detected as a marker of the envelope protein. (D) Localization of AtYLMG1-1 examined by immunofluorescence microscopy. Isolated chloroplasts from the wild type were immunostained with the anti-AtYLMG1-1 antibodies. The green fluorescence indicates the localization of AtYLMG1-1 and the red shows the chlorophyll fluorescence. Bar = 5 μm. (E) Relationship between AtYLMG1-1 puncta and chloroplast nucleoids. Isolated chloroplasts were immunostained with the anti-AtYLMG1-1 antibodies and counterstained with DAPI. The red indicates the localization of AtYLMG1-1 and the blue is DAPI fluorescence showing the localization of DNA. A merged image is also shown. Arrowheads indicate the overlap between the AtYLMG1-1 puncta and nucleoids. Bar = 5 μm.