Jocelyn Bédard

Functional Specialization amongst the Arabidopsis Toc159 Family of Chloroplast Protein Import Receptors

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis

β-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other β-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.

Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip).

Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (ΔSti1), a second lacking a central region, including the putative TPR domain (ΔTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the ΔSti1 and ΔTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites.

A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis.

SUMMARY The sensitive to freezing2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.

In Vivo Analyses of the Roles of Essential Omp85-Related Proteins in the Chloroplast Outer Envelope Membrane

Two different, essential Omp85 (Outer membrane protein, 85 kD)-related proteins exist in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD), encoded by atTOC75-III; and OEP80 (Outer Envelope Protein, 80 kD), encoded by AtOEP80/atTOC75-V. The atToc75-III protein is closely related to the originally identified pea (Pisum sativum) Toc75 protein, and it forms a preprotein translocation channel during chloroplast import; the AtOEP80 protein is considerably more divergent from pea Toc75, and its role is unknown. As knockout mutations for atTOC75-III and AtOEP80 are embryo lethal, we employed a dexamethasone-inducible RNA interference strategy (using the pOpOff2 vector) to conduct in vivo studies on the roles of these two proteins in older, postembryonic plants. We conducted comparative studies on plants silenced for atToc75-III (atToc75-III↓) or AtOEP80 (AtOEP80↓), as well as additional studies on a stable, atToc75-III missense allele (toc75-III-3/modifier of altered response to gravity1), and our results indicated that both proteins are important for chloroplast biogenesis at postembryonic stages of development. Moreover, both are important for photosynthetic and nonphotosynthetic development, albeit to different degrees: atToc75-III↓ phenotypes were considerably more severe than those of AtOEP80↓. Qualitative similarity between the atToc75-III↓ and AtOEP80↓ phenotypes may be linked to deficiencies in atToc75-III and other TOC proteins in AtOEP80↓ plants. Detailed analysis of atToc75-III↓ plants, by electron microscopy, immunoblotting, quantitative proteomics, and protein import assays, indicated that these plants are defective in relation to the biogenesis of both photosynthetic and nonphotosynthetic plastids and preproteins, confirming the earlier hypothesis that atToc75-III functions promiscuously in different substrate-specific import pathways.

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope

Chloroplast chaperone Hsp93 associates with the inner envelope membrane via interaction with the Tic110 translocon and likely functions in quality control but not in import propulsion of translocating preproteins. The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.

Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana

Homologs of a bacterial β-barrel protein, Omp85, ubiquitously exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Those in non-photosynthetic bacteria and mitochondria are responsible for β-barrel protein sorting to the outer membranes, and thus are essential for viability of the organisms. There are two distinct Omp85 homologs in chloroplasts of the model plant, Arabidopsis thaliana. One of them, Toc75, functions as the main protein import translocation channel, and was shown to be indispensable from a very early stage of embryogenesis. By contrast, the role of another homolog, OEP80, remains elusive. Recently, we showed that disruption of the OEP80 gene causes embryo abortion in A. thaliana at a stage later than that affected by TOC75 knockout. This indicates that the two chloroplastic Omp85 homologs are both essential for viability of plants from very early stages of development, but may have distinct functions. Defining the functional and evolutionary relationships of Toc75 and OEP80 by further studies should advance our understanding of the importance of plastids during embryogenesis, as well as that of the molecular details of plastid biogenesis. Addendum to: Patel R, Hsu SC, Bédard J, Inoue K, Jarvis P. The Omp85-related chloroplast outer envelope protein, OEP80, is essential for viability in Arabidopsis. Plant Physiol 2008; 148:235-45; DOI: 10.1104/pp.108.122754.

Green light for chloroplast outer-membrane proteins

Most chloroplast proteins are encoded in the cell nucleus, translated in the cytosol, and targeted to the organelle in a post-translational manner. Our understanding of how proteins are targeted to the outer envelope membrane of chloroplasts has been improved with the identification of a specific soluble sorting factor.

Suppressors of the Chloroplast Protein Import Mutant tic40 Reveal a Genetic Link between Protein Import and Thylakoid Biogenesis

Genetic analyses identify the chloroplast proteins ALB4 and STIC2 as coactors in a specialized thylakoid protein targeting pathway, and a link between envelope translocation and thylakoid targeting. To extend our understanding of chloroplast protein import and the role played by the import machinery component Tic40, we performed a genetic screen for suppressors of chlorotic tic40 knockout mutant Arabidopsis thaliana plants. As a result, two suppressor of tic40 loci, stic1 and stic2, were identified and characterized. The stic1 locus corresponds to the gene ALBINO4 (ALB4), which encodes a paralog of the well-known thylakoid protein targeting factor ALB3. The stic2 locus identified a previously unknown stromal protein that interacts physically with both ALB4 and ALB3. Genetic studies showed that ALB4 and STIC2 act together in a common pathway that also involves cpSRP54 and cpFtsY. Thus, we conclude that ALB4 and STIC2 both participate in thylakoid protein targeting, potentially for a specific subset of thylakoidal proteins, and that this targeting pathway becomes disadvantageous to the plant in the absence of Tic40. As the stic1 and stic2 mutants both suppressed tic40 specifically (other TIC-related mutants were not suppressed), we hypothesize that Tic40 is a multifunctional protein that, in addition to its originally described role in protein import, is able to influence downstream processes leading to thylakoid biogenesis.

A Ycf2-FtsHi heteromeric AAA-ATPase complex is required for chloroplast protein import

A protease-originated membrane-bound AAA-ATPase is involved in preprotein translocation across the inner envelope membrane of chloroplasts as an essential import motor. Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.