| Bacterial expression of two rhomboid protease homologs from Arabidopsis thaliana and assessing their activity with the plastid envelope protein, Tic40 (2007) | |||||||||||
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| The regulated intramembrane proteolysis mechanism remains to be studied in great detail in the plant system, while it is well studied in animals. However, the recent elucidation of a putative rhomboid-like protease gene in the Arabidopsis thaliana genome presents the possible link of this mechanism. The key to finding this link would be in confirming activity of the rhomboid-like protease on a native substrate in the plant cell. In this current work, two Arabidopsis rhomboid protease homologs were cloned into an E. coli bacterial expression system in the presence of Tic40, an integral plastid envelope protein. This in vitro assay presents a simple method of affirming the possibilty that Tic40 is a substrate of at least one of the plant rhomboid protease homologs. Developing this assay involves numerous checks at both the cloning and over-expression steps meant to ensure that this assay could provide significant evidence of protease activity on Tic40. The results from the development of this assay indicate that there is likely a proteolytic interaction between rhomboid protease and Tic40. They also lend support to our initial speculation that rhomboid protease cleaves in the insoluble portion of a protein substrate such as within the transmembrane region. It is hypothesized that the plant rhomboid protease functions in the processing of a Tic40 preprotein to its mature form for its correct localization to the inner membrane of the chloroplast envelope. The results provide a promising beginning to a new paradigm in plant cell biology research, especially in understanding the biological mechanisms involved in protein trafficking and localization by post-translational proteolytic processing. | |||||||||||
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