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Hromatke, Geraldine (2015): Identifizierung und Charakterisierung der mitochondrialen Translokationspore Tim23.2 als Calmodulin-bindendes Protein. Dissertation, LMU München: Fakultät für Biologie



In order to react to changes within their environment, plants developed a specific signaling network that enables the cells to convert external stimuli including light, abiotic and biotic stress as well as hormones into cellular signals. One example is the influx of calcium, a second messenger stored in apoplasts or internal reservoirs, into the cytosol. This causes changes in the calcium-ion-concentration within the cell that are recognized by specific sensors including Calmodulin and lead to the induction of a cellular signal response. Calcium signals do not only occur in the cytosol, but also appear within the nucleus, chloroplasts, mitochondria as well as peroxisoms (Bachs et al. 1992, Chigri et al. 2005, Kuhn et al. 2009, Dolze et al. 2013). The import of nuclear encoded proteins into the mitochondria is regulated by calcium and Calmodulin at level of the TIM23- and TIM22-complex. This study identified atTim23.2, the pore-forming component of the TIM23-complex, as a Calmodulin-binding protein. Pull-down-assays using Calmodulin-agarose revealed a specific and calcium-dependent binding. Furthermore, in silico analysis identified two potential Calmodulin-binding domains (CaMBD). Topology studies of atTim23.2 demonstrated that the proposed N-terminal CaMBD is located within the intermembrane space, the binding region within the first loops is located in the matrix of the mitochondria. Moreover, a topology of four transmembran domains of the protein could be shown. The recently in the mitochondria identified Calmodulin-like protein CML30 appeared to be a potential binding partner for atTim23.2. CML30 could be indeed detected in the intermembrane space of the mitochondria, but a direct interaction of the two proteins could not have been detected so far. Furthermore, using the split-ubiquitin system proved the ability of atTim23.2 to dimerize which might be responsible for the regulation of opening and closing of the importpore as it was already shown in S.cerevisiae. However, a correlation between the two functions of atTim23.2 to bind Calmodulin as well as to dimerize could not have been confirmed, yet. Nevertheless, the regulation of the pore via the calcium/Calmodulin signaling network could connect the import process of matrix proteins with the stress regulation of the cell.