Genetic and Biochemical Analyses of the Arabidopsis atToc90 Protein

Chloroplasts are photosynthetic organelles in plant and algal cells that capture sunlight energy to form energy-rich molecules that are the basis for almost all life. Chloroplast development requires more than 3000 different proteins, most of which are encoded by nuclear DNA. Thus, chloroplasts must import most of their proteins from the cytosol. They are surrounded by a double membrane called the envelope. Embedded in the envelope are the TOC and TIC complexes (translocon at the outer and inner envelope membrane of the chloroplast, respectively), which mediate protein import into the organelle. Several components of the TOC and TIC complexes have been identified. One example is the receptor Toc159, which in the model plant Arabidopsis thaliana has four isoforms: atToc159, atToc132, atToc120 and atToc90. It is known that atToc159 supports accumulation of photosynthetic proteins, while atToc132 and atToc120 support the import of non-photosynthetic, housekeeping proteins. However, the role of atToc90 remains uncertain. I investigated the function of atToc90 genetically by studying a series of Arabidopsis toc90 double and triple mutants, and by overexpressing atToc90 in mutants lacking other receptor isoforms. This work suggested limited functional redundancy between atToc90 and other TOC receptors (most notably, atToc159). By tagging TOC receptors known to act in each of the photosynthetic and non-photosynthetic import pathways, I was able to purify different TOC complexes from transgenic plants using tandem affinity purification (TAP). This indicated that atToc90 is present promiscuously in both atToc159- and atToc132/120-containing TOC complexes. Publicly available Affymetrix microarray data suggested a role for atToc90 during senescence. Thus, I investigated whether toc90 knockout mutants display any differences from wild type regarding leaf senescence. Indeed, some defects were observed, suggesting a role for atToc90 in the biochemical changes that occur in chloroplasts during leaf senescence.