Funtionelle Charakterisierung von mTERF-homologen mitochondriellen Proteinen in pflanzlichen Systemen am Beispiel des kernkodierten Faktors MOC1 aus C. reinhardtii
Final Report Abstract
The mitochondrial transcription termination factor (mTERF) MOC1 was identified by the characterization of the knock-out stm6, which was among mutants generated within a forward genetics approach aiming at the identification of genes implicated in light-acclimation processes. At the beginning of the funding period only the physiological consequences of a MOC1 deletion had been described partially and the precise molecular function of MOC1 was largely unknown. The major intention of my project was to get insights into the molecular mechanisms of MOC1 action. Vital tools for such a project are antisera specifically recognizing the studied protein and an expression system allowing to obtain large amounts of pure protein for various in vitro studies. The MOC1 antiserum was successfully used in immunoblot studies, which confirmed that MOC1 is indeed a mitochondrial protein and that the recent gene annotation predicts the size of the mature protein correctly. Confirmation the mitochondrial localization was crucial, since other plant mTERFs were already shown to localize to the plastid. Although MOC1 turned out be prone to inclusion body formation a systematic optimization of the expression conditions by use of different solubility tags and strains eventually yielded in the successful purification of recombinant MOC1 from E. coli. The recombinant MOC1 binds to double-stranded DNA and EMSA experiments demonstrated that MOC1 possesses a sequence-specific binding activity. A systematic search for specific binding sites within the mitochondrial genome was conducted and at least three different binding sites located downstream of nd1 were identified by EMSA studies with overlapping mtDNA probes. The mutant displays a consistent down-regulation of the nd1 mRNA, which is a mitochondrial gene encoding a subunit essential for the assembly of respiratory complex I. This finding nicely correlated to a new phenotypic characteristic observed for stm6 within the grant period. Growth under strictly heterotrophic conditions is severely impaired in the mutant and complementation with nuclear expression vectors containing the coding sequence of MOC1 rescue the ability to grow normally on acetate in the dark. Among the deliverables of the fellowship period are MOC1 knock-down strains generated by artificial microRNA-induced gene silencing. These strains display the main phenotypical features of stm6 and will therefore represent important controls for future experiments intending to analyse the consequences of reduced MOC1 availability. In addition to the knockdown strains several strains showing recovered dark growth were obtained by transformation of stm6 with a MOC1-cDNA expressed under the control of different strong constitutive promoters. Both systems can be applied to study the effects of MOC1 expression on the expression levels of certain mitochondrial mRNAs. In conclusion the fellowship was used to obtain important tools for the analysis of MOC1 function. The protein is targeted to the mitochondrion, binds to double-stranded DNA in a specific manner and is obviously a key factor involved in the expression regulation of certain mitochondrial genes including nd1.
Publications
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(2009) Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas. Proc. Natl. Acad. Sci. USA 106: 13290-13295
Wobbe, L., Blifernez, O., Schwarz, C., Mussgnug, J.H., Nickelsen, J. & Kruse, O.
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(2009) Improvement of light to biomass conversion by de-regulation of light-harvesting protein translation in Chlamydomonas reinhardtii. J. Biotechnol. 142: 70-77
Beckmann, J., Lehr, F., Finazzi, G., Hankamer, B., Posten, C., Wobbe, L. & Kruse, O.
