A Molecular-Genetic Study of the Arabidopsis Toc75 Gene Family

Abstract

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 splicejunction 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.The majority of plastid proteins are translated on cytosolic ribosomes and subsequently imported into plastids (Keegstra and Cline, 1999;Chen et al., 2000;Hiltbrunner et al., 2001a;Jarvis and Soll, 2001;Jarvis and Robinson, 2004). An amino-terminal transit peptide directs each of these proteins specifically to the plastid. Upon arrival in the stroma, the transit peptide is cleaved and the mature protein is either folded into its final conformation or targeted to another compartment of the plastid (Keegstra and Cline, 1999;Jarvis and Robinson, 2004). Preproteins are translocated through the plastid double membrane envelope by two membrane-bound protein complexes: the translocon at the outer envelope membrane of chloroplasts (Toc), and the translocon at the inner envelope membrane of chloroplasts (Tic).Components of the Toc complex include Toc34, Toc75, and Toc159, which were first identified in pea (Pisum sativum) using biochemical approaches (Hirsch et al., 1994;Perry and Keegstra, 1994;Schnell et al., 1994;Seedorf et al., 1995;Tranel et al., 1995). These three proteins make up the core Toc complex, which was recently characterized with respect to structure and component stoichiometry (Schleiff et al., 2003b). Toc34 and Toc1...

The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis

“…2, A and B), strongly supporting the notion that the homozygous genotypes were responsible for seed abortion. Identification of two independent mutant alleles that give rise to the same phenotype is widely accepted as proof of a causal relationship (Sjö gren et al, 2004;Baldwin et al, 2005;Puyaubert et al, 2008). Thus, our data provide very strong evidence of an essential role for AtOEP80.…”

“…By contrast, atToc75-III is synthesized as a larger precursor with a bipartite transit peptide, like psToc75, and is highly expressed (Inoue and Keegstra, 2003;Baldwin et al, 2005). Disruption of atTOC75-III with T-DNA insertions arrested embryo development at a very early stage (the two-cell stage; Baldwin et al, 2005;Hust and Gutensohn, 2006). These results indicate that atToc75-III is the most direct functional ortholog of psToc75.…”

“…Previous work demonstrated that atTOC75-I is a pseudogene and that atTOC75-IV encodes a truncated protein that lacks a transit peptide and is expressed at very low levels . By contrast, atToc75-III is synthesized as a larger precursor with a bipartite transit peptide, like psToc75, and is highly expressed (Inoue and Keegstra, 2003;Baldwin et al, 2005). Disruption of atTOC75-III with T-DNA insertions arrested embryo development at a very early stage (the two-cell stage; Baldwin et al, 2005;Hust and Gutensohn, 2006).…”

Chloroplast Omp85 proteins change orientation during evolution

“…13) and the chloroplast proteins Toc75-III and Toc75-V/Oep80 (14). Remarkably, all three are essential as mutations such as sam50 in yeast and toc75-III or toc75-V are lethal (15)(16)(17).…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…The Arabidopsis genome encodes two paralogs of Toc34 (atToc33 and atToc34); Gutensohn et al , 2000 ), and four paralogs of Toc159 (atToc159, atToc132, atToc120 and atToc90; Bauer et al , 2000 ;Hiltbrunner et al , 2004 ;Kubis et al , 2004 ) and Toc75 (atToc75-III, atToc75-IV, atToc75-I and atToc75V/ atOep80) (Eckart et al , 2002 ;Baldwin et al, 2005). The discovery of multiple arrays of isoforms in Arabidopsis promises to make the functional characterization of each individual component of the chloroplast import system challenging.…”