Mitochondria are subcellular organelles with their own genome and expression system, including translation machinery to make proteins. Several independent studies have shown that translation is an essential regulatory step in expression of the plant mitochondrial genome. Thus, the study of mitochondrial translation seems to be crucial for the comprehension of plant mitochondrial biogenesis and maintenance. In organello protein synthesis in isolated mitochondria is a direct method to visualize the translational products of this organellar genetic system. In this method, highly purified, functional mitochondria synthesize proteins in the presence of radiolabeled amino acids, such as methionine, and an energy regeneration system. The labeled, newly synthesized polypeptides are separated by SDS-polyacrylamide gel electrophoresis and are detected by autoradiography. Here we describe the detailed protocol for in organello labeling of translation products that was optimized for mitochondria isolated from rosette leaves and liquid culture seedlings of Arabidopsis thaliana plants.
Keywords: Arabidopsis; Autoradiography; In Organello Protein Synthesis; Mitochondria; SDS-PAGE; [35S]-Methionine Incorporation.
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