In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue

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Abstract
Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 h. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [35S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro. Analysis, by SDS-polyacrylamide gel electrophoresis and autoradiography, showed that isolated mitochondria synthesized about 15 polypeptides in the MW range 8000-55,000. As aging proceeded, significant changes were observed in the relative rates of labeling of 3 out of the 8 major polypeptides synthesized by mitochondria in vitro.