Protein Import

Background

Chloroplasts are descendent from an ancient photosynthetic prokaryote (an ancestor of extant cyanobacteria) that entered into an endosymbiotic relationship with an early eukaryotic progenitor over a billion years ago. While present-day plastids retain a functional, endogenous genome, the evolutionary transfer of genetic material to the cell nucleus has meant that most (>90%) of the ~3000 genes required for their biogenesis are now nucleus-encoded. Organellar gene displacement led to the need for sophisticated mechanisms to import proteins from the cytosol, where they are made, across the double membrane system, or envelope, that surrounds each plastid. Protein targeting to chloroplasts is of vital importance for plants, since these organelles account for the majority of protein in leaves, and are the unique site for the energy-capturing process, photosynthesis. All proteins destined for interior locations within plastids are synthesized as precursor proteins (preproteins) that carry cleavable, amino-terminal extensions called transit peptides. Transit peptides direct proteins into plastids via a specific protein import pathway, and are removed following transfer across the envelope. Import is mediated by the coordinate actions of translocon complexes in the outer and inner envelope membranes called TOC and TIC (Translocon at the Outer / Inner envelope membrane of Chloroplasts).
 

The Protein Import Apparatus

Figure 1: The Chloroplast Protein Import Apparatus

Biochemical studies using isolated pea chloroplasts have led to the identification of several components of the TOC and TIC complexes  (Figure 1). The three, major outer envelope components of the import apparatus (Toc159, Toc34 and Toc75; numbers indicate molecular weights in kD) were identified by their association with translocon-engaged preproteins. Toc159 and Toc34 interact with preproteins at a very early stage and are believed to act as preprotein receptors. They are both GTPases, accounting for the fact that early stages of import require GTP. Toc75, on the other hand, forms the outer membrane channel through which translocation occurs. The role of a fourth TOC component, Toc64, remains to be elucidated.

Several putative or actual components of the inner envelope complex have also been identified (i.e., Tic20, Tic21, Tic22, Tic32 Tic40, Tic55, Tic62 and Tic110), although in many cases specific roles have not been defined. The Tic22 protein may be involved in coordinating the activities of the TOC and TIC complexes, and/or in preprotein recognition at the inner envelope, while Tic20, Tic21 and Tic110 have all been proposed to play roles in forming the inner envelope channel. Tic110 acting together with Tic40 and molecular chaperones (Hsp93 and Hsp70) also forms part of the "import motor" that drives protein translocation. Tic32, Tic55 and Tic62 are putative regulatory components, linking import rates with redox poise within the photosynthetic apparatus. Molecular chaperones associated with both envelope membranes maintain preproteins in an unfolded, import-competent state and, as already mentioned, provide the driving force for translocation.
 

Figure 2: Model for Chloroplast Protein Import
 

Chloroplast Protein Import in Arabidopsis

Historically, chloroplast protein import was studied in vitro using isolated pea chloroplasts and biochemical techniques. These studies led to the identification of several putative components of the import apparatus, as already mentioned. More recently, genes encoding homologues of these pea proteins have been identified by the Arabidopsis genome sequencing project. Interestingly, multiple forms of Toc159 and Toc34 were identified in Arabidopsis. The Arabidopsis Toc34 homologues are called atToc33 and atToc34 (the "at" prefix simply denotes species of origin: A. thaliana), while the Toc159 homologues are called atToc159, atToc132, atToc120 and atToc90. The existence of multiple TOC protein isoforms, and the fact that the genes are differentially regulated, led to the proposal that there are multiple, different translocon complexes in Arabidopsis with different substrate (preprotein) specificities. Operation of different import pathways, associated with these different translocon complexes, may serve to prevent damaging competition effects between highly-abundant preproteins (e.g., photosynthesis-related proteins) and less-abundant preproteins (e.g., house-keeping proteins); alternatively, they may play a role in the differentiation of different plastid types (e.g., chloroplasts vs. non-green plastids such as amyloplasts and chromoplasts).
 

The identification of Arabidopsis TOC/TIC genes, and the demonstrated utility of Arabidopsis molecular-genetic techniques for studying chloroplast protein import in vivo, have together led to the establishment of Arabidopsis as a new and versatile model system for studying chloroplast protein import. The first mutant with a defect in a translocon component to be identified was the Arabidopsis plastid protein import 1 (ppi1) mutant (Jarvis et al., 1998, Science 282:100-103). This mutant is null for atToc33 and has a striking yellow-green phenotype (Figure 3). Isolated ppi1 chloroplasts import photosynthetic preproteins with reduced efficiency, leading to the notion that the atToc33 receptor isoform acts in an import pathway with preference for highly-abundant, photosynthetic preproteins (Kubis et al., 2003, Plant Cell 15: 1859-1871). An atToc159 null mutant (ppi2) has also been identified, by other researchers, and its analysis led to the conclusion that the atToc159 receptor isoform is similarly specialized for photosynthetic preproteins, and that it acts together with atToc33 in the same import pathway. Work in the Jarvis laboratory is currently focused on the identification and characterization of new Arabidopsis import mutants, using a variety of forward- and reverse-genetic strategies, and on the more detailed characterization of existing Arabidopsis mutants.
 

Figure 3: Visible Phenotype of the ppi1 Mutant