Protein Import

Current Work

Figure 3: Visible Phenotype of the ppi1 Mutant
 

Characterization and Exploitation of Existing and New Knockout Mutants

Figure 4: Analysis of atTic40 Deletion Mutants

The Arabidopsis ppi1 mutant was the first protein import mutant to be identified (Figure 3). It is null for the Toc34 receptor isoform, atToc33, and has a recessive yellow-green phenotype (Jarvis et al., 1998, Science 282:100-103). Detailed analysis of the ppi1 mutant has provided significant insights into the roles of the receptor GTPases in chloroplast protein import. In view of the informative nature of the analyses performed using the ppi1 mutant, we and others employed reverse genetics to identify new Arabidopsis knockout mutants lacking many of the other known translocon components: sequencing of the Arabidopsis genome revealed genes encoding homologues of each TOC/TIC protein originally identified in pea, and this information enabled the identification of new Arabidopsis mutants (most are T-DNA insertion mutants). Thus, today, ppi1 is just one of a relatively large number of Arabidopsis import mutants that have been identified (for a complete list, see Jarvis, 2008, New Phytol. 179: 257-285). These various knockout mutants are being characterized in detail in order to shed light on the operation of individual components within the import mechanism. In addition, double (and triple, etc.) TOC/TIC mutants are being isolated and studied to address functional interactions between the proteins, and to assess the absolute importance of translocon component functions represented by multiple homologous genes.

The knockout mutants are also being used as genetic backgrounds in which to assess the effects of point mutations (e.g., Aronsson et al., 2010, Plant J. 63: 297-311), domains deletions (e.g., Figure 4), or domain-swaps (e.g., Bédard et al., J. Biol. Chem. 282: 21404-21414) on the functionality of various components of the import machinery. Additionally, the mutants represent an invaluable resource for biochemical studies, as they allow one to replace the native copy of a specific component with a tagged form of the protein (e.g., using the tandem affinity purification tag, or TAP tag). Transgenic plants expressing a tagged protein are useful for various analyses, including protein complex purification.

A major limitation of the reverse-genetic approach described above is that it cannot be used to identify new components of the import apparatus. In view of the fact that there are many unanswered questions concerning the chloroplast protein import mechanism, it seems likely that additional translocon proteins, or regulatory factors, remain to be discovered. Because molecular and biochemical approaches have already been employed extensively, we are using a range of novel, forward-genetic strategies to identify new components or regulators of the translocation complexes.

Arabidopsis ppi1 and tic40 are both viable mutants that lack known, well-characterized components of the import machinery (atToc33 and atTic40, respectively) leading to specific defects in the import mechanism. Thus, these mutant genotypes (along with others that are similarly under investigation) represent ideal starting points for the genetic dissection of the import mechanism by suppressor analysis. Because there are fundamental differences between the two mutants (in relation to function, localization, essentiality, etc.), we reasoned (correctly) that the targets of the respective suppressor screens would be distinct. Two mutagenic strategies have been employed for the generation of suppressor mutants: ethyl methanesulfonate (EMS) mutagenesis, and activation tagging. Suppressors of ppi1 and tic40 have been identified, extensively characterized, and the genes responsible for the suppressor phenotypes have been cloned. Current work aims to elucidate the mechanism of suppression in each case.