- Members of the Preprotein and Amino Acid Transporter Family Constitute Components of Novel Protein Import Pathways into Chloroplasts (2011)
- In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membranes. Translocons at the outer and inner chloroplast envelope membranes, called TOC and TIC, were identified that mediate the import of proteins. N-terminal transit peptides essential for import of the protein precursors are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursors would enter the chloroplast through the same, jointly acting TIC/TOC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. One example is the chloroplast envelope quinone oxidoreductase homologue, ceQORH. This protein is imported into the inner plastid envelope membrane by a TIC/TOC-independent pathway and without any proteolytic cleavage. In the present study 5 proteins were shown to interact with ceQORH during its import and were designated as ceQORH translocon components (QTC). One of these proteins, QTC24 (also called HP20), is a member of the PRAT family comprising preprotein and amino acid transporters found in chloroplasts, mitochondria and free-living bacteria. Different expression patterns and localization of PRAT proteins suggest that they are functionally diverse beyond their role in protein translocation. QTC24/HP20 is located in the outer plastid envelope membrane of chloroplasts where it establishes a hydrophilic translocation pore. Thus, chloroplasts contain besides TOC75 and OEP16-1 a third translocation channel component in their outer envelope membrane that functions in import of transit sequence-less inner envelope proteins. In vitro import into chloroplasts of corresponding isolated A. thaliana knock-out mutants revealed that the lack of HP20 could not be replaced by its close relative HP22. Athp20 plants had no phenotype when grown under standard green house conditions. However, minor defects during the very early stage of greening of etiolated seedlings were observed as the expression of mainly plastid-encoded proteins was delayed. These effects could be interpreted in terms of an impaired amino acid import at this stage of development. A second protein of the PRAT family, HP30, was further subject of this work. However, its role remains unclear at the moment. Isolated homozygous A. thaliana knock-out mutants of HP30 did not reveal any phenotype under the growth conditions analysed in this work. The preliminary investigation of stable RNA silencing mutants indicated that the function of HP30 and its close relative HP30-2 is important during the early stages of seedling development. Young leaves of respective mutant plants exhibited a chlorotic phenotype. A further member of the PRAT family is OEP16-1 that was initially identified as amino acid-selective protein channel. Other studies revealed its role as translocation pore for the PORA precursor. Analysis of the corresponding A. thaliana knock-out mutant to dissect these two mutually not exclusive functions has led to the description of different phenotypes. During a re-screen of the original seed stock, four independent OEP16-1-deficient mutant lines were isolated that exhibited different cell death properties. Two mutants contained elevated amounts of free protochlorophyllide (Pchlide) in darkness that was caused by a defect in the Pchlide-dependent import of PORA. Etiolated seedlings of these lines died after light exposure due to the production of singlet oxygen. The two other mutants did not accumulate excessive amounts of free Pchlide and greened normally. Two of the four mutant lines with seemingly no correlation between the lack of PORA and cell death were analysed in more detail in this thesis. Moreover, a complemented Atoep16-1 mutant that re-expressed functional OEP16-1 protein was shown to restore the wild-type phenotype including PORA import that prevented the accumulation of an excess of free Pchlide and singlet oxygen production upon light exposure of dark-grown seedlings.