Chloroplasts are a hallmark feature of plant cells and the sites of photosynthesis – the process in which plants harness the energy in sunlight for their own needs. The first chloroplasts arose when a photosynthetic bacterium was engulfed by another host cell, and most of the original bacterial genes have been transferred to the host cell’s nucleus during the evolution of land plants. As a result, modern chloroplasts need to import the thousands of proteins encoded by these genes from the rest of the cell.

The chloroplast protein import system relies on a protein transporter in the chloroplast membrane that evolved from a family of bacterial transporters. However, the bacterial transporters were initially involved in protein export, and it was not known how the activity of these transporters adapted to move proteins in the opposite direction.

Paila et al. set out to better understand the chloroplast protein import system and produced mutated forms of the transporter in the model plant Arabidopsis thaliana. These experiments revealed that a part of the transporter that is conserved in many other organisms, the “protein transport associated domains”, has been adapted for three key roles in protein import. First, this part of the transporter interacts with the other components of the import system that make the transporter more selective and control which direction the proteins are transported. Second, the domains interact with proteins during transport to help move them across the chloroplast membrane. Finally, the domains recruit other molecules called chaperones, which stop the protein from aggregating or misfolding during the transport process. These activities are similar to those for the bacterial export transporters, but clearly evolved to allow transport in the opposite direction – that is, to import proteins into chloroplasts.

The next challenges are to explain how proteins destined for chloroplasts are recognized and transported through the chloroplast’s membrane.

The biogenesis and function of plastids are reliant on the coordinate expression and selective import of >2500 different nucleus-encoded proteins (Ling and Jarvis, 2015, Shi and Theg, 2013). The major pathway for protein import involves the synchronized action of two multi-protein membrane translocons, the TOC (translocon at the outer envelope membrane of chloroplasts) and the TIC (translocon at the inner envelope membrane of chloroplasts) complexes (Paila et al., 2015, Lee et al., 2014), which reside within the chloroplast envelope. TOC and TIC complexes contain selective protein conducting channels, which align via physical association between the complexes to provide simultaneous membrane translocation of preproteins from the cytoplasm to the plastid interior (Paila et al., 2015, Shi and Theg, 2013, Ling and Jarvis, 2015). The selectivity and directionality of protein import is controlled by sequential interactions between the targeting determinants within the cleavable, N-terminal transit peptides of preproteins and binding sites within the TOC and TIC complexes (Li and Teng, 2013), as well as import-associated molecular chaperones (Flores-Pérez and Jarvis, 2013, Paila et al., 2015, Schwenkert et al., 2011).

The evolution of the TOC and TIC system during endosymbiosis required the adaptation or acquisition of protein conducting channels, receptors and molecular chaperones at both envelope membranes to provide a unidirectional conduit for the transport of nuclear encoded polypeptides into the organelle. Protein transport across the outer envelope membrane is mediated by a β-barrel membrane protein, Toc75, which is proposed to constitute the major component of the TOC import channel (Hinnah et al., 2002). Toc75 assembles with two integral membrane receptor GTPases, Toc159 and Toc34, which recognize plastid preproteins in the cytoplasm and control access of preproteins to the Toc75 channel via their intrinsic GTPase activities (Chang et al., 2012, Kessler and Schnell, 2009, Kessler and Schnell, 2004). In addition to its role as the TOC channel, Toc75 is proposed to participate in the targeting and insertion of membrane-anchored outer membrane proteins, including the TOC GTPases (Wallas et al., 2003).

Toc75 is encoded by a single gene in Arabidopsis (TOC75-III, AT3G46740) and null mutants are embryo lethal (Jackson-Constan and Keegstra, 2001, Baldwin et al., 2005, Hust and Gutensohn, 2006). Knockdown of TOC75-III expression by RNAi (Huang et al., 2011) and a hypomorphic toc75-III mutant (Stanga et al., 2009) both result in pale phenotypes and growth defects, consistent with the role of Toc75 in chloroplast biogenesis. Based on these studies, Toc75 appears to have evolved to perform multiple functions in protein import, in the assembly of TOC complexes, and in the biogenesis of the outer membrane (Richardson et al., 2014).

Toc75 belongs to the outer membrane protein of 85 kDa (Omp85) superfamily of β-barrel integral membrane proteins (Hsu and Inoue, 2009). Omp85 proteins are exclusively localized to the outer membranes of Gram-negative bacteria, mitochondria and plastids (Inoue and Potter, 2004, Schleiff et al., 2011, Voulhoux and Tommassen, 2004), and perform major roles in protein transport and the insertion and assembly of outer membrane proteins (Voulhoux and Tommassen, 2004, Schleiff et al., 2011). For example, BamA (β-barrel Assembly Machinery protein A) and Sam50 (Sorting and Assembly Machinery 50 kDa), two members of the Omp85 family, function in outer membrane biogenesis in Gram-negative bacteria and mitochondria, respectively, by facilitating the folding and insertion of β-barrel proteins at the outer membrane from the periplasmic or intermembrane space (Voulhoux et al., 2003, Noinaj et al., 2013, Schleiff and Soll, 2005). The structural similarity of Toc75 to these conserved membrane transporters led to the hypothesis that Toc75 evolved from an ancestral Omp85 gene and was adapted during endosymbiosis of the cyanobacterial ancestor to function as the protein import channel in the plastid outer envelope (Day et al., 2014, Topel et al., 2012, Bredemeier et al., 2007, Gentle et al., 2005).

Toc75 exhibits two main structural features characteristic of Omp85 superfamily members. The N-terminal ~30 kDa region of Toc75 contains three repeats of POTRA (polypeptide-transport associated) domains, each characterized by a β₁α₁α₂β₂β₃ secondary structural motif (Clantin et al., 2007, Koenig et al., 2010, Paila et al., 2015). The ~45 kDa C-terminal region is predicted to contain 16 membrane-spanning β-strands and constitute the membrane-integrated β-barrel (Paila et al., 2015, Day et al., 2014). While there is significant evidence that the C-terminal β-barrel domain of Toc75 acts as a component of the translocon channel for protein translocation at the outer membrane (Hinnah et al., 1997), the functions of the N-terminal POTRA domains have not been clearly defined.

In the case of BamA, specific POTRA repeats are indispensable for the insertion of β-barrel proteins at the E. coli outer membrane (Browning et al., 2013). The POTRA domains of BamA extend into the periplasm, where they interact with other components of the BAM complex (Bam B-E), a major periplasmic chaperone, SurA (Bennion et al., 2010), and nascent outer membrane proteins (Kim et al., 2007, Noinaj et al., 2015). Structural studies of BamA also suggest that specific POTRA repeats interact with the C-terminal domain to act as a possible gate to the membrane channel of the β-barrel (Noinaj et al., 2013, Noinaj et al., 2015, Bakelar et al., 2016). In another example, deletion of a major portion of the POTRA domain of Sam50 in yeast mitochondria inhibits growth (Habib et al., 2007), and subsequent studies provided evidence that the domain interacts with β-barrel precursors to promote their release from the SAM complex and insertion in the outer membrane from the intermembrane space (Kutik et al., 2008, Stroud et al., 2011). The studies of BamA and Sam50 are consistent with models in which the POTRA repeats are assembled into multi-functional cassettes to mediate protein-protein interactions required for the assembly and specific targeting function of the membrane biogenesis machinery (Koenig et al., 2010).

The relationship between Toc75 and other Omp85 superfamily members raises interesting evolutionary and mechanistic questions of how the structural and functional features of the conserved POTRA and β-barrel domains of Toc75 have adapted during endosymbiosis to constitute the protein import channel. Structural and electrophysiological studies of Toc75 demonstrate that the protein forms a cation-selective channel with a pore size sufficient to transport unfolded polypeptide substrates (Hinnah et al., 1997; 2002). In vitro pull-down experiments with recombinant POTRA domains of Toc75 suggest that they can interact with the TOC GTPases and chloroplast preproteins (Ertel et al., 2005). This has led to the proposal that they function in TOC complex assembly, interactions with the TOC GTPases, preprotein recognition, or to provide a chaperone-like activity for the preprotein during membrane translocation (Sánchez-Pulido et al., 2003, Ertel et al., 2005). In this study, we combine molecular genetic and biochemical analyses to examine the roles of the POTRA domains in protein import and membrane targeting in chloroplasts. We demonstrate that POTRA domains contribute essential functions to the Toc75 channel. Furthermore, expression of Toc75 lacking one or more of the three POTRA repeats leads to dominant negative phenotypes in Arabidopsis thaliana. Expression of specific deletion mutants alters the size and distribution of the TOC complexes and disrupts preprotein import into the organelle. Biochemical analyses indicate that the Toc75 POTRAs interact specifically with chloroplast preproteins and two proposed chaperones of the intermembrane space, Tic22-III and Tic22-IV. Our results demonstrate multiple roles for the POTRAs in protein import, including TOC complex assembly, preprotein translocation and the recruitment of chaperones to facilitate transport across the intermembrane space.

The number and function of POTRA domains in Omp85 family members vary, but in all systems, they mediate interactions with transport substrates and/or factors that assist with Omp85 protein function (Jacob-Dubuisson et al., 2009). POTRA domains are defined by repeats of conserved β₁α₁α₂β₂β₃ secondary structural motifs (Clantin et al., 2007, Koenig et al., 2010, Paila et al., 2015), and the variation in primary structure within the repeats contributes to the functional diversification and adaptation of family members for specific roles in the transport and insertion of nascent polypeptides at the outer membrane in bacteria and mitochondria (Sanchez-Pulido et al., 2003, Koenig et al., 2010). Our analysis of Toc75 is consistent with a multifunctional cassette model for POTRA domains, and highlights the unique adaptation of the Toc75 POTRA domains to mediate interactions that are essential for assembly of the core TOC components and the transport of polypeptides across the outer membrane.

We took advantage of the observation that expression of Toc75ΔP1, Toc75ΔP1-2 and Toc75ΔP1-3 in the heterozygous toc75III-1 background resulted in plants with pale phenotypes and significant growth defects (Figure 1C). The phenotypes were dependent on the expression levels of the deletion constructs, and severe effects were observed even at levels of Toc75ΔP1, Toc75ΔP1-2 and Toc75ΔP1-3 that were far less than equimolar to endogenous native Toc75 (Figure 1C). Given that TOC translocons are large multimeric complexes containing component ratios estimated at 4:4:1 or 3:3:1 (Toc75:Toc34:Toc159), the insertion of defective POTRA deletion subunits into these complexes likely interferes with the coordinate functions of the native subunits.

Toc75ΔP1 and Toc75ΔP1-2 were targeted, integrated and fully processed at the outer membrane (Figure 6A and B), with their POTRA domains extending into the intermembrane space (Figure 6), which is consistent with the topology of native Toc75 and all other known Omp85 family members (Sanchez-Pulido et al., 2003, Stroud et al., 2011). This indicates that the POTRA domains do not play essential roles in targeting and membrane insertion of Toc75 itself. The orientation of the Toc75 POTRA domains in the intermembrane space also is consistent with the observation that they specifically interact with Tic22 isoforms, proteins localized to the intermembrane space (Figure 9). A separate study using N-terminal fusions of Toc75 to self-assembling split GFP (saGFP) concluded that the N-terminus of Toc75 was localized in the cytoplasm when transiently expressed in Arabidopsis protoplasts (Sommer et al., 2011). However, the saGFP-Toc75 constructs used in the study were not shown to complement the lethal phenotype of toc75-III-1 mutants, and therefore, it is unclear if the fusions assumed a functional topology in the outer membrane.

Our studies of protein import into isolated chloroplasts from the TOC75ΔP1#2 and TOC75ΔP1-2#2 plants demonstrate that the POTRA deletions resulted in a significant reduction in the rate of protein import into chloroplasts (Figure 3). The analysis of saturation-binding experiments under low [ATP] and [GTP] revealed that the decrease in import was due to the reduction in the total number of functional import sites rather than a significant reduction in the affinity of TOC complexes for the preprotein (Figure 5). Furthermore, the levels of TOC components were unchanged or increased in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants (Figure 7), suggesting that the decreased import capacity resulted from defective TOC function and not from decreases in the overall levels of the core TOC proteins.

Our data reveal an interesting function for the POTRA domains in TOC complex assembly that accounts, at least in part, for the decreased levels of functional TOC complexes observed in TOC75ΔP1#2 and TOC75ΔP1-2#2 chloroplasts. Expression (Figure 7) and 2D blue-native PAGE (Figure 8) analyses revealed significant changes in the levels and assembly of the core TOC components, which likely results in a population of defective or non-functional translocons. Toc75ΔP1 appeared to retain the ability to assemble with both Toc33 and Toc159 GTPase receptors into 1.3 MDa and 440 kDa complexes (Kikuchi et al., 2006), suggesting that POTRA1 is not required for stoichiometric assembly of TOC complexes. However, Toc75ΔP1-2 was only detected in novel complexes that migrated between 200–880 kDa. A major fraction of Toc33, but not native Toc75 or Toc159, exhibited a coincident broad range of distribution with Toc75ΔP1-2 (Figure 8B). Together with the observation that Toc75ΔP1-2 co-immunoprecipitated with Toc33, this suggests that Toc75ΔP1-2 is able to associate normally with Toc33, but not Toc159, evidenced by the lack of Toc159 in the 200–880 kDa complexes in TOC75ΔP1-2 chloroplasts. This implicates POTRA-2 alone, or in combination with POTRA-1, in interactions between Toc75 and Toc159, which is a requirement for proper TOC complex assembly and function.

The role of Toc75 POTRAs in TOC assembly is reminiscent of the role of the five repeat POTRA domains of the BamA in the assembly of the BAM complex at the outer membrane. POTRA 5 of BamA interacts with the essential periplasmic lipoprotein, BamD, to form the core of the complex (Malinverni et al., 2006, Sklar et al., 2007, Wu et al., 2005). The core complex recruits BamC and E (Albrecht and Zeth, 2011, Kim et al., 2007), and POTRA 2–4 of BamA interact with BamB (Kim et al., 2007, Kim and Paetzel, 2011, Noinaj et al., 2011, Dong et al., 2012) to constitute the fully functional BAM complex. Despite the conservation of functional domains, Toc75 appears to be unique amongst Omp85 family members because it evolved to mediate protein import into plastids from the cytoplasm. The reversal of the transport activity relative to the bacterial and mitochondrial Omp85 family members occurred with the diversification of Toc75 from the cyanobacterial Omp85 ancestor and was driven by the association of Toc75 with the Toc33 and Toc159 receptors. Toc33 and Toc159 provide the GTP-dependent driving force for the unidirectional transport of preproteins from the cytoplasm into the organelle. The Toc33-Toc75-Toc159 core is a universal feature of the plant lineage, and the adaptation of POTRAs to facilitate these interactions was, therefore, a key event in the evolution of TOC complexes.

Our data also indicate that disruption of Toc75 function in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants perturbs the known coordinate expression of the TOC components (Sun et al. 2001), resulting in significant over-expression of Toc33 relative to Toc75 and Toc159. The major portion of the excess Toc33 migrated at ~70 kDa on blue-native PAGE of TOC75ΔP1#2 and TOC75ΔP1-2#2 chloroplasts and was not associated with the other TOC components (Figure 8B). The excess Toc33 could contribute to the phenotypes observed in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants if it interacted with preproteins and interfered with import. However, our quantitative binding data indicate that energy-independent and energy-dependent bindings are reduced in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants (Figures 4 and 5), arguing against preprotein binding to excess Toc33 as a significant factor in TOC75ΔP1#2 and TOC75ΔP1-2#2 phenotypes.

We also hypothesize that the Toc75 POTRA domains play a direct role in protein translocation across the outer membrane by selectively binding to chloroplast preproteins in the intermembrane space (Figure 9). This proposal is consistent with our observation that the isolated POTRA domains interact with a chloroplast preprotein, preSSU, in a transit-peptide and dose-dependent manner, and previous data demonstrating an interaction between the N-terminal region of Toc75 and preproteins targeted to chloroplasts (Ertel et al., 2005). Several possibilities exist for the role of the POTRA-transit peptide interaction during protein import. Toc75 crosslinks to the transit peptides of preproteins during initial binding as well as during later stages of membrane translocation (Ma et al., 1996), suggesting that the POTRA domains could contribute to

preprotein recognition in conjunction with the TOC GTPases and/or stabilize preprotein binding to the translocon upon initial translocation through the Toc75 membrane pore. The latter possibility appears to be most likely, considering the localization of the POTRA domains in the intermembrane space, and the observation that Toc75ΔP1 or Toc75ΔP1-2 did not impact energy-independent binding of preprotein to chloroplasts (Figures 4 and 5).

The substrate-binding activity of POTRA domains has precedence in other Omp85 family members. The POTRA domain of BamA binds nascent β-barrel proteins as they emerge into the periplasmic space from the Sec translocon in the bacterial inner membrane, suggesting that the BamA POTRAs form a platform for the polypeptide substrate to assume the necessary secondary structure required to initiate membrane insertion (Gatzeva-Topalova et al., 2008, Kim et al., 2007, Knowles et al., 2008). POTRA-5, located most adjacent to the β-barrel, is proposed to play an essential role in assisting the insertion of the substrate into the membrane directly or in coordination with the β-barrel and the oligomeric BAM complex (Sinnige et al., 2015, Noinaj et al., 2013). The single POTRA of Sam50 β-barrel component of the mitochondrial SAM machinery also is proposed to assist in release of the β-barrel substrate into the mitochondrial outer membrane (Kutik et al., 2008, Stroud et al., 2011).

Recent crystal structures of BamA showed two different orientations of its POTRA domains, leading to the proposal that the domains are involved in a gating mechanism for regulating access of the substrate to the the β-barrel membrane insertase (Noinaj et al., 2013, Bakelar et al., 2016). A crystal structure and molecular dynamics simulations of POTRA domains of OMP85 from Anabaena revealed a similar conserved feature in these proteins (Koenig et al., 2010), and mutations in the unique loop region of the POTRA domains adjacent to the β-barrel in the cyanobacterial protein influenced the pore properties of the β-barrel (Koenig et al., 2010, Ertel et al., 2005). It is tempting to speculate that a similar gating mechanism might function at Toc75, thereby providing a mechanism by which transit peptide binding at TOC complexes controls the outer membrane channel by triggering reorientation of the POTRA domains.

Finally, we also demonstrate specific interactions of the Toc75 POTRA domains with two homologous import components of the intermembrane space, Tic22-III and Tic22-IV. Tic22 from pea was previously shown to covalently cross-link to preprotein import intermediates (Kouranov et al., 1998), and proteins with significant similarity to Tic22 from apicoplasts of Toxoplasma gondii and Plasmodium falciparum have been shown to exhibit chaperone activity (Glaser et al., 2012). Interestingly, a Tic22-like protein from cyanobacteria was shown to covalently cross-link with an Omp85 family member of the cyanobacterial outer membrane (Tripp et al., 2012), providing a precedent for the interaction of Omp85 family members with the Tic22 family of putative chaperones, and its potential conservation from the cyanobacterial endosymbiont. This interaction is also reminiscent of the interaction between the BamA POTRAs and a periplasmic chaperone, SurA, during β-barrel insertion at the outer membrane (Bennion et al., 2010).

The significant increase in the levels of Tic22 isoforms in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants is consistent with a functional association between the Toc75 POTRA domains and the Tic22 chaperones and could represent a response to a disruption in chaperone-preprotein interactions during membrane translocation. To date, a chaperone activity for Tic22-III and Tic22-IV in plants has not been investigated, and it will be of great interest to understand how the Toc75 POTRA domains might coordinate interactions between preproteins and the putative chaperone to facilitate preprotein transit through the intermembrane space and engagement of the TIC complex.

Our analysis of Toc75 is consistent with models in which the POTRA domains play versatile roles in TOC assembly and function, which account for the decreased levels of functional TOC complexes seen in TOC75ΔP1#2 and TOC75ΔP1-2#2 chloroplasts. As is the case in other Omp85 family members, each domain is likely to contribute individual activities, but also work in association with the other domains to mediate assembly of the translocon complex and coordinate preprotein translocation. In contrast to bacterial and mitochondrial outer membranes, chloroplasts contain two Omp85 family members: Toc75 and OEP80 (Outer Envelope Protein 80) (Hsu and Inoue, 2009). Similar to Toc75, OEP80 is essential in Arabidopsis (Patel et al., 2008) and is predicted to contain three N-terminal soluble POTRA domains and a C-terminal β-barrel (Hsu and Inoue, 2009, Hsu et al., 2012). OEP80 is not detected in isolated oligomeric TOC complexes, and it has been proposed to function in β-barrel protein biogenesis in a manner analogous to Sam50 and BamA (Eckart et al., 2002). Although phylogenetic analyses indicated that Toc75 and OEP80 are more closely related to each other than either is to other bacterial Omp85 family members, distinct sequence signatures have been identified in both the POTRA and β-barrel domains of the chloroplast proteins to account for evolutionary divergence of these two β-barrel channels (Day et al., 2014). The evolution of an import apparatus to mediate the transport of nucleus-encoded preproteins into plastids was an essential element of endosymbiosis. Our data are consistent with the divergent evolution of Toc75 and OEP80 from the ancestral cyanobacterial OMP85 gene to retain the pathway for β-barrel protein biogenesis, while providing a novel pathway for protein import into the organelle.

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Author details

1. Yamuna D Paila

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   YDP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Lynn GL Richardson

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   LGLR, This author was added because the first author of this manuscript (Paila) took parental leave with her first child right after we received the comments from reviewers. Dr. Richardson completed the experimental and textual revisions of the manuscript. Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Hitoshi Inoue

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   HI, Conception and design, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Elizabeth S Parks

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   ESP, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. James McMahon

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   JM, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Kentaro Inoue

   Department of Plant Sciences, University of California, Davis, United States

   Contribution

   KI, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Danny J Schnell

   Department of Plant Biology, Michigan State University, East Lansing, United States

   Contribution

   DJS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   dschnell@cns.msu.edu

   Competing interests

   The authors declare that no competing interests exist.

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