A molecular-genetic study of the Arabidopsis Toc75 gene family

Toc75 ( translocon at the outer envelope membrane of chloroplasts, 75 kD) is the protein translocation channel at the outer envelope membrane of plastids and was first identified in pea ( Pisum sativum) using biochemical approaches. The Arabidopsis ( Arabidopsis thaliana) genome contains three Toc75- related sequences, termed atTOC75- I, atTOC75- III, and atTOC75- IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTOC75- III is strongly regulated and at its highest level in young, rapidly expanding tissues. By contrast, atTOC75- IV is expressed uniformly throughout development and at a much lower level than atTOC75- III. The third sequence, atTOC75- I, is a pseudogene that is not expressed due to a gypsy/ Ty3 transposon insertion in exon 1, and numerous nonsense, frame- shift, and splice-junction mutations. The expressed genes, atTOC75- III and atTOC75- IV, both encode integral envelope membrane proteins. Unlike atToc75- III, the smaller atToc75- IV protein is not processed upon targeting to the envelope, and its insertion does not require ATP at high concentrations. The atTOC75- III gene is essential for viability, since homozygous atToc75- III knockout mutants ( termed toc75- III) could not be identified, and aborted seeds were observed at a frequency of approximately 25% in the siliques of self- pollinated toc75- III heterozygotes. Homozygous toc75- III embryos were found to abort at the two- cell stage. Homozygous atToc75- IV knockout plants ( termed toc75- IV) displayed no obvious visible phenotypes. However, structural abnormalities were observed in the etioplasts of toc75- IV seedlings and atTOC75- IV overexpressing lines, and toc75- IV plants were less efficient at deetiolation than wild type. These results suggest some role for atToc75- IV during growth in the dark.