Fig. 2.
Intracellular localization of OOP (At5g65620) and CyOP (At5g10540). (A) Fluorescence imaging of the full-length OOP fused to GFP with a mitochondrial cherry (mit-cherry) and chloroplastic cyan blue (chloro-CFP) marker in A. thaliana suspension-cultured cells. Mitochondria (m) and plastids (p) are indicated by arrows. (Scale bar: 20 µm.) (B) Detection of FLAG-tagged OOP in cytosol (Cy), mitochondria (M), and chloroplasts (C) isolated from stable A. thaliana transformants. (C) Fractionation of mitochondria and chloroplasts isolated from stable A. thaliana transformants expressing OOP-FLAG. Presence of FLAG was detected in total mitochondria (M), mitoplasts (Mp), inner membrane (IM), and matrix (Ma) fractions as well as in total chloroplasts (C), stroma (S), and thylakoids (T). (D) Analysis of proteolytic maturation of OOP in mitochondria and chloroplasts. In vitro synthesized and radioactively labeled precursor of OOP (pre-OOP) was resolved on SDS/PAGE alongside purified OOPΔ1–82 and total mitochondria, (M) and chloroplasts (C) isolated from wild-type A. thaliana plants. OOP was detected either by autoradiography or by immunodetection. The asterisk indicates the precursor form. (E) Fluorescence imaging of full-length CyOP fused to GFP and cytosolic, mitochondrial, and chloroplastic marker proteins fused to RFP in A. thaliana suspension-cultured cells. (Scale bar: 20 µm.) (F) Detection of FLAG-tagged CyOP in cytosol (Cy), mitochondria (M), and chloroplasts (C) isolated from stable A. thaliana transformants. Antibodies used in B–D and F were anti-FLAG, anti-AtPreP (mitochondrial matrix and chloroplastic stroma), anti-SHMT (serine hydroxymethyltransferase, mitochondrial matrix), anti-Tim17.2 (mitochondrial inner membrane), anti-PsbA (D1 protein, thylakoid membrane), anti-Tim9 (mitochondrial intermembrane space), anti-RbcL (chloroplastic stroma), anti-OOP, and anti-UGPase (cytosolic marker). WB, Western blot analysis.