Substrate selection by the FtsH protease

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The essential AAA-type protease FtsH of Escherichia coli has protein quality control and regulatory functions. We are studying the role of FtsH in controlling the heat shock response by degradation of the heat shock sigma factor RpoH (s32). Multiple factors (DnaK, DnaJ, RNA polymerase) are involved in this process. Two strategies have been used to generate random mutations in rpoH: (i) linker insertion mutagenesis, and (ii) error-prone mutagenesis in a bacterial two-hybrid system designed to select for protease cleavage sites. Conformational changes and protein-protein interactions of RpoH proteins derived from the first approach are going to be studied by biophysical techniques. Initial results from the two-hybrid screening suggest that an important turnover element of RpoH is located around amino acid 47. Additional mutations in this region will be constructed by site-directed mutagenesis. Degradation of the RpoH variants will be analyzed in vivo and in vitro. Their interaction with partner proteins involved in FtsH-mediated proteolysis will be examined. To extent our studies on substrate selection by the FtsH protease, we plan to exploit the two-hybrid technology for other FtsH substrates (l cII and cIII). Moreover, a genetic screen to identify the turnover element in the most important FtsH substrate LpxC (EnvA) will be established.