Arabidopsis Genes Encoding Components of the Chloroplastic Protein Import Apparatus

Abstract

The process of protein import into plastids has been studied extensively using isolated pea (Pisum sativum) chloroplasts. As a consequence, virtually all of the known components of the proteinaceous apparatus that mediates import were originally cloned from pea. With the recent completion of the Arabidopsis genome sequencing project, it is now possible to identify putative homologs of the import components in this species. Our analysis has revealed that Arabidopsis homologs with high sequence similarity exist for all of the pea import complex subunits, making Arabidopsis a valid model for further study of this system. Multiple homologs can be identified for over one-half of the components. In all but one case it is known that more than one of the putative isoforms for a particular subunit are expressed. Thus, it is possible that multiple types of import complexes are present within the same cell, each having a unique affinity for different chloroplastic precursor proteins, depending upon the exact mix of isoforms it contains. Sequence analysis of the putative Arabidopsis homologs for the chloroplast protein import apparatus has revealed many questions concerning subunit function and evolution. It should now be possible to use the genetic tools available in Arabidopsis, including the generation of knockout mutants and antisense technology, to address these questions and learn more about the molecular functions of each of the components during the import process.The availability of the sequence for the entire genome of Arabidopsis allows a detailed analysis of all the genes involved in a particular biological process, regardless of the plant species in which the system was first identified. One such process is the import of cytoplasmically synthesized precursor proteins into chloroplasts. Most of the current information regarding this process, including the identification of components of the import apparatus that mediates it, has come from biochemical studies in pea (Pisum sativum; Fig. 1; Chen and Schnell, 1999; Keegstra and Cline, 1999; Keegstra and Froehlich, 1999;May and Soll, 1999; Schleiff and Soll, 2000). From these studies it has been determined that nuclear-encoded, chloroplastlocalized enzymes are synthesized in the cytoplasm as precursors containing an N-terminal transit peptide not seen in the mature protein within the chloroplast (for review, see Bruce, 2000). A precursor protein initially interacts with a complex located within the outer membrane of the chloroplast envelope that consists of at least three subunits: translocon at the outer envelope membrane of chloroplasts (Toc) 159, Toc75, and

The Arabidopsisppi1Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins[W]

“…An alternative explanation is that reduced overall levels of a functionally redundant atToc33 ϩ atToc34 pool in ppi1 affects the import of those preproteins with the lowest affinity for the import apparatus most strongly. However, such a model does not explain why Arabidopsis has retained multiple isoforms of certain components of the import apparatus, such as Toc34 and Toc159, and only single isoforms of others, such as Tic110 and Tic40 (Jackson-Constan and Keegstra, 2001). Furthermore, this model is inconsistent with the fact that ppi1 significantly affected the import of preSSU ( Figure 5A).…”

“…The plastome-encoded Rubisco large subunit gene, rbcL, is one obvious exception. Also of interest is the observation that all of the protein targeting-related genes were upregulated in ppi1, except for the gene that encodes Tic20-I, which is a putative channel component of the inner envelope membrane (Jackson-Constan and Keegstra, 2001). Similarly, genes that encode several different molecular chaperones, some of which have been implicated in chloroplast protein import Nielsen et al, 1997;Jackson-Constan and Keegstra, 2001), also were upregulated in ppi1 (Table 2; see also supplemental data online).…”

“…For example, there are two Toc34-related genes in Arabidopsis, atTOC33 and atTOC34 (Jarvis et al, 1998;Gutensohn et al, 2000), and four Toc159-related genes, atTOC159 , atTOC132 , atTOC120 , and atTOC90 (Bauer et al, 2000;Hiltbrunner et al, 2001a). There also are multiple Toc75-related sequences in Arabidopsis, but some other components are encoded by just a single gene (Jackson-Constan and Keegstra, 2001). The reason for this Toc component multiplicity in Arabidopsis is unclear, but it has been proposed that different Toc isoforms have different preprotein recognition specificities (Jarvis et al, 1998).…”

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

“…Ectopic expression of atToc34 rescues ppi1 indicating that atToc34 and atToc33 are functionally equivalent in vivo (Jarvis et al, 1998). There are four homologues of pea Toc159 in Arabidopsis, atToc159, -132, -120 and -90 (Hiltbrunner et al, 2001a;Jackson-Constan and Keegstra, 2001). The initial characterization of the last, atToc90, is presented in this study.…”

“…We report on the characterization of a sixth, and last, member of the Toc-family of GTP-binding proteins, a group of proteins involved in the early stages of chloroplast protein import (Hiltbrunner et al, 2001a;Jackson-Constan and Keegstra, 2001). Based on its sequence similarity and molecular mass, we have designated the protein encoded by the ORF in Arabidopsis EST-clone H4C12T7 atToc90.…”