Jarvis Lab

Protein Import


Two thirds of primary production on the planet depends on photosynthesis by land ecosystems. Thus, most of our food and most of the oxygen in the air that we breath derives ultimately from plants. Chloroplasts are the cellular organelles responsible for photosynthesis in plants, and so it is evident that these organelles are of vital importance, not only to plants but also to humans and most other organisms.

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 action 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

Biochemical studies using isolated pea chloroplasts have led to the identification of several components of the TOC and TIC complexes  (figure 2). 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, is a beta-barrel protein and forms the outer membrane channel through which preprotein 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, although in many cases their 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 (e.g., Hsp93 and Hsp70) is thought to form part of an "import motor" that drives preprotein translocation. Tic32, Tic55 and Tic62 are putative regulatory components, linking import rates with redox poise within the photosynthetic apparatus. Molecular chaperones associated with the envelope membranes maintain preproteins in an unfolded, import-competent state and, as already mentioned, provide the driving force for translocation.

Chloroplast protein import can be divided into three stages based on the energetic requirements (determined in vitro) for progression through each step (figure 3). The first stage (energy-independent binding) is reversible, and does not require ATP or GTP. The second stage (early import intermediate formation) requires low concentrations of ATP (100 μM) and GTP, and is irreversible. Proteins at this stage are inserted across the outer envelope membrane and are in contact with the TIC machinery, but remain remain partially exposed at the organellar surface. The third stage of import (complete translocation) requires higher concentrations of ATP (1 mM), in the chloroplast interior or stroma, but no GTP. Preproteins are processed to yield their mature forms inside the organelle, and import can then be considered complete. The GTP requirement during early import intermediate formation is attributed to the Toc159 and Toc34 GTPases, which play critical roles in preprotein recognition, while the bulk of the ATP consumption is attributed to molecular chaperones in the stromal import motor.


Protein Import Research in the Jarvis Lab

Historically, chloroplast protein import was studied in vitro using isolated pea chloroplasts and biochemical techniques. Such studies led to the identification of several putative components of the import apparatus, as summarized above. Subsequently, genes encoding homologues of these pea proteins were identified by various genome sequencing projects. Work in the Jarvis laboratory has sought to complement the biochemically-derived data from pea by bringing to bear the unique set of advantages afforded by the model plant, Arabidopsis thaliana.

Chloroplast Protein Import in Arabidopsis

Arabidopsis thaliana (thale cress) has been adopted at research institutions across the globe as the model organism of choice for molecular, genetic, genomic, biochemical, physiological, bioinformatic and systems studies on diverse aspects of plant biology (http://www.arabidopsis.org/). This is because the advantages associated with studying Arabidopsis are many. For example: the Arabidopsis genome has been completely sequenced, and so researchers know exactly which genes are present; the plant is easy to transform, and so transgenic experiments are routine; extremely large collections of “knockout” mutants are available publicly, meaning that mutants in almost any gene can be obtained; because Arabidopsis is so widely studied across the globe, well-established protocols are available for almost any conceivable type of experiment; the plant is small, grows rapidly, and produces large quantities of seed, making it easy to manage in a laboratory environment.

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 Toc34-type protein called atToc33 (see below), and has a striking yellow-green phenotype (figure 4). Isolated ppi1 mutant chloroplasts import photosynthesis-related preproteins with reduced efficiency, leading to the notion that atToc33 acts in an import pathway with preference for highly-abundant, photosynthetic preproteins (Kubis et al., 2003, Plant Cell 15: 1859-1871).

Interestingly, multiple genes coding for the protein import receptors, Toc159 and Toc34, were identified in Arabidopsis (and in other plants). 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 receptor isoforms, and the fact that the genes are differentially regulated, led to the proposal that there are multiple, different translocon complexes with different client (preprotein) specificities. Operation of different import pathways, as enabled by 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), or play a role in the differentiation of different plastid types (e.g., chloroplasts vs. non-green plastids such as amyloplasts and chromoplasts).

Characterization and Exploitation of Knockout Mutants

As mentioned above, the Arabidopsis ppi1 mutant was the first protein import mutant to be identified. 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 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 list, see Jarvis, 2008, New Phytol. 179: 257-285). These various knockout mutants have been (and continue to be) 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 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), domain deletions (e.g., figure 5), or domain-swaps (e.g., Bédard et al., J. Biol. Chem. 282: 21404-21414) in relation to 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.

Forward-Genetic Screens to Identify Novel Import-Related Components

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. Ongoing work aims to elucidate the mechanism of suppression in each case.

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 Last updated: Jan 2014
 Paul Jarvis