Investigation of folding and degradation of in vitro synthesized mutant proteins in mitochondria.

Abstract

AbstractMissense mutations and one-amino acid deletions or insertions affect in a majority of cases folding and/or the stability of the native structure (1–3). Investigation of the biogenesis of the mutant protein after synthesis of the fulllength polypeptide is therefore an essential starting point to evaluate the effect of a given mutation. Many disease-causing missense mutations cause retention of partly folded and misfolded intermediates in complexes with molecular chaperones or their premature degradation and these are hallmarks for folding deficiencies. For mitochondrial proteins, the use of in vitro transcription/translation followed by addition of isolated mitochondria presents a simple and efficient way to investigate the biogenesis of the mutant protein inside the organelle. The advantage of the method is that the protein of interest can be selectively labeled in vitro and subsequently its biogenesis can be investigated in its normal environment in the organelle without the need to use immunological methods to distinguish it from the other proteins. Mitochondria can easily be isolated from animal tissues, but can also be isolated from cultured cells (4).The mitochondrial protein is first synthesized in a coupled in vitro transcription/translation system using reticulocyte lysate. Subsequently, the synthesized polypeptide is added to freshly prepared mitochondria from rat liver. After a short import step at low temperature (20°C) the mitochondria are shifted to higher temperature and samples are taken at different time intervals.KeywordsShake Water BathNative PageTranslational Initiation SiteSonication Water BathPhosphorimager AnalysisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.