Jarvis Lab - Chloroplast Protein Import

Background

Photosynthesis is the process that harnesses sunlight to energize life on Earth, fixing carbon and generating oxygen as a by-product. In plants, photosynthesis takes place in tiny subcellular structures (organelles) called chloroplasts [1]. Given that all of our food, and the oxygen in the air we breathe, derives ultimately from photosynthesis, it is evident that these organelles are vitally important, not only to plants but to humans and essentially all life on Earth.

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 chloroplasts retain a functional endogenous genome, the evolutionary transfer of genetic material has meant that most (>90%) of the ~3000 genes required for chloroplast biogenesis are now encoded in the nucleus. Because nucleus-encoded proteins are synthesized in the cytosol, this relocation of organellar genes led to the need for sophisticated mechanisms to deliver proteins from the cytosol into the chloroplast across the double-membrane system (or envelope) that surrounds each organelle [2,3].

Proteins destined for interior locations within chloroplasts (or other types of plastid) are synthesized as precursor proteins (preproteins), each one carrying a cleavable, amino-terminal extension called a transit peptide. The transit peptide is essentially an address tag, directing the preprotein into the organelle via a specific protein import pathway; and it is removed following the completion of transfer across the envelope. This protein import process is mediated by the coordinate action of multiprotein translocon complexes located in the outer and inner envelope membranes called TOC and TIC (for Translocon at the Outer / Inner envelope membrane of Chloroplasts) [2,3].

The Chloroplast Protein Import System

Numerous studies conducted by different groups led to the identification of many components of the TOC and TIC complexes (figure 2) [3]. The three major components of the outer-membrane TOC complex (Toc159, Toc33 and Toc75; numbers indicate molecular weights in kDa) were identified by their association with translocon-engaged preproteins. Toc159 and Toc34 interact with preproteins at an 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 β-barrel protein (a member of the Omp85 superfamily) and forms the outer-membrane channel through which preprotein translocation occurs.

Several putative or actual components of the inner envelope TIC complex have also been identified, although in many cases their specific roles have not been defined [3]. The intermembrane space (IMS)-spanning protein, Tic236, and the chaperone protein, Tic22, may be involved in coordinating the activities of the TOC and TIC complexes, or guiding preproteins to the TIC machinery. The Tic20 protein lies at the core of a 1-MDa complex also containing the chloroplast-encoded subunit Tic214 and several other proteins; this complex may form the main, channel-forming translocon. Two other proteins with large domains facing the chloroplast interior (the stroma) are Tic110 and the co-chaperone Tic40, and these may act downstream to coordinate the activities of stromal molecular chaperones (e.g., Hsp93 and Hsp70), acting as part of an import motor that drives preprotein translocation. Alternatively, a 2-MDa ATPase complex containing several FtsH-related subunits may perform the motor role. Upon completion of import, the transit peptide is cleaved off by the stromal processing peptidase (SPP) (figure 2).

Preproteins are translocated through the import machinery in unfolded conformation, N-terminus first, and molecular chaperones act to maintain them in an unfolded, import-competent conformation [2]. 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 partially exposed at the organellar surface. The third stage of import (complete translocation) requires higher concentrations of ATP (1 mM) in the 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 Toc33 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 our 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 [4,5].

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 [6]. This is because the advantages associated with studying Arabidopsis are many. For example, it was the first plant to have its genome completely sequenced, meaning that researchers have long known exactly which genes are present; it is easy to genetically transform, and so transgenic experiments are routine; there are multiple, large collections of “knockout” mutants which are available publicly, meaning that mutants in almost any gene can readily be obtained and studied; well-established protocols are available for almost any conceivable type of experiment with Arabidopsis because the plant is so widely studied across the globe; and, lastly, the plant is easy to manage in a laboratory setting because it is small, grows rapidly, and produces large quantities of seed.

The identification of Arabidopsis TOC/TIC genes, and the demonstrated utility of Arabidopsis molecular-genetic techniques for studying chloroplast protein import, together led to the establishment of Arabidopsis as a new and versatile model system in the import field. The first mutant with a defect in a translocon component to be identified was the Arabidopsis plastid protein import 1 (ppi1) mutant [7]. This mutant is null for the import receptor, Toc33, and has a striking yellow-green phenotype (figure 4). Chloroplasts of the ppi1 mutant import photosynthesis-related preproteins with reduced efficiency, leading to the notion that Toc33 acts in an import pathway with preference for highly-abundant, photosynthetic preproteins [8].

Interestingly, multiple genes coding for the protein import receptors, Toc159 and Toc33, were identified in Arabidopsis (and in other plants). The Arabidopsis Toc33 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 led to the hypothesis that there are multiple, different translocon complexes with different client (preprotein) specificities (figure 5) [1-3]. 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., housekeeping 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 atToc33 receptor isoform, and has a recessive yellow-green phenotype [7]. 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 this mutant, we and others employed reverse genetics to identify new Arabidopsis knockout mutants lacking other translocon components: the Arabidopsis genome sequence revealed genes encoding homologues of each TOC/TIC protein, enabling the identification of new Arabidopsis mutants (most are T-DNA insertion mutants). Thus, today, ppi1 is just one of a large number of Arabidopsis import mutants that have been identified (for a list, see ref. 2). These various knockout mutants have been (and continue to be) characterized in detail in order to shed light on the operation of the import system. In addition, double (and triple, etc.) TOC/TIC mutants are studied to address functional interactions between the proteins, and to assess the importance of translocon component functions encoded by multiple homologous genes.

The knockout mutants are also being used as genetic backgrounds in which to assess the effects of point mutations [9], domain deletions (e.g., figure 6), or domain-swaps (e.g., ref. 10) on the function of import machinery components. The mutants also 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 a tandem affinity purification tag or other epitope tags). Transgenic plants expressing a tagged protein are useful for various analyses, including affinity purification for structural analyses.

Forward-Genetic Approaches to Identify Novel Import-Related Components

A significant limitation of the reverse-genetic approach described above is that it does not lend itself to the identification of new components of the import apparatus. Given that there are many unanswered questions concerning the chloroplast protein import system, it is likely that additional components or regulatory factors remain to be uncovered. Thus, to complement the molecular and biochemical approaches that have been successfully employed previously, we are using a range of novel, forward-genetic strategies to deliver fresh insights. Two examples of this type of approach are described below.

Arabidopsis ppi1 and tic40 are both viable mutants that lack known, well-characterized components of the import machinery (atToc33 and atTic40, respectively), with consequent defects in the import process. Thus, these mutant genotypes (along with others that are similarly under investigation) are ideal starting points for the forward-genetic dissection of the import process, through suppressor analysis. Because there are fundamental differences between the two mutants (in relation to function, localization, essentiality, etc.), the targets of the respective suppressor screens were expected to be distinct – and so it proved [11,12]. Moreover, different mutagenic strategies can be employed to further extend the range of possible outcomes. Our ongoing work aims to elucidate the mechanism of suppression in each case, and makes use of the mutants as tools to elucidate different aspects of chloroplast protein homeostasis. Of note, the ppi1 suppressor screen led to the identification of a chloroplast-localized ubiquitin E3 ligase, SUPPRESSOR OF PPI1 LOCUS1 (SP1), which plays a central role in the regulation of chloroplast protein import pathways (figure 8); and, ultimately, to the discovery of the chloroplast-associated protein degradation (CHLORAD) system (figure 9) [13].

References
1. Jarvis, P., López-Juez, E. (2013) Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14: 787-802.
2. Jarvis, P. (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley Review). New Phytol. 179: 257-285.
3. Sun, Y., Jarvis, R.P. (2023) Chloroplast proteostasis: import, sorting, ubiquitination, and proteolysis. Annu. Rev. Plant Biol. 74: 259-283.
4. Jarvis, R.P., ed. (2011) Chloroplast Research in Arabidopsis: Methods and Protocols, Vols. I and II. Methods in Molecular Biology, Vols. 774 and 775. Humana Press, Totowa, NJ, USA.
5. Jarvis, P. (2019) Chloroplast research methods: probing the targeting, localization and interactions of chloroplast proteins. J. Vis. Exp. doi:10.3791/59935.
6. http://www.arabidopsis.org/
7. Jarvis, P., Chen, L.-J., Li, H.-m., Peto, C., Fankhauser, C. and Chory, J. (1998) An Arabidopsis mutant defective in the chloroplast general protein import apparatus. Science 282: 100-103.
8. Kubis, S., Baldwin, A., Patel, R., Razzaq, A., Dupree, P., Lilley, K., Kurth, J., Leister, D. and Jarvis, P. (2003) The Arabidopsis ppi1 mutant is specifically defective in the expression, chloroplast import and accumulation of photosynthetic proteins. Plant Cell 15: 1859-1871.
9. Aronsson, H., Combe, J., Patel, R., Agne, B., Martin, M., Kessler, F. and Jarvis, P. (2010) Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency. Plant J. 63: 297-311.
10. Bédard, J., Kubis, S., Bimanadham, S. and Jarvis, P. (2007) Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hip. J. Biol. Chem. 282: 21404-21414.
11. Ling, Q., Huang, W., Baldwin, A., Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659.
12. Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell 29: 1726-1747.
13. Ling, Q., Broad, W., Trösch, R., Töpel, M., Demiral Sert, T., Lymperopoulos, P., Baldwin, A. and Jarvis, R.P. (2019) Ubiquitin-dependent chloroplast-associated protein degradation in plants. Science 363: eaav4467.


	Jarvis Lab	Protein Import