Chloroplast import motor subunits FtsHi1 and FtsHi2 are located on opposite sides of the inner envelope membrane.

Protein import into chloroplasts is powered by ATP hydrolysis in the stroma. Establishing the identity and functional mechanism of the stromal ATPase motor that drives import is critical for understanding chloroplast biogenesis. Recently, a complex consisting of Ycf2, FtsHi1, FtsHi2, FtsHi4, FtsHi5, FtsH12, and malate dehydrogenase was shown to be important for chloroplast protein import, and it has been proposed to act as the motor driving protein translocation across the chloroplast envelope into the stroma. To gain further mechanistic understanding of how the motor functions, we performed membrane association and topology analyses on two of its subunits, FtsHi1 and FtsHi2. We isolated cDNA clones encoding FtsHi1 and FtsHi2 preproteins to perform in vitro import experiments in order to determine the exact size of each mature protein. We also generated antibodies against the C-termini of the proteins, i.e., where their ATPase domains reside. Protease treatments and alkaline and high-salt extractions of chloroplasts with imported and endogenous proteins revealed that FtsHi1 is an integral membrane protein with its C-terminal portion located in the intermembrane space of the envelope, not the stroma, whereas FtsHi2 is a soluble protein in the stroma. We further complemented an FtsHi1-knockout mutant with a C-terminally tagged FtsHi1 and obtained identical results for topological analyses. Our data indicate that the model of a single membrane-anchored pulling motor at the stromal side of the inner membrane needs to be revised and suggest that the Ycf2-FtsHi complex may have additional functions.

TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import.

SignificanceAlthough plastid division is critical for plant development, how components of the plastid division machinery (PDM) are imported into plastids remains unexplored. A forward genetic screen to identify suppressors of a crumpled leaf (crl) mutant deficient in plastid division led us to find dominant gain-of-function (GF) mutations in TIC236, which significantly increases the import of PDM components and completely rescues crl phenotypes. The defective plastid division phenotypes in crl and tic236-knockdown mutants and CRL-TIC236 association in a functional complex indicate that the CRL-TIC236 module is vital for plastid division. Hence, we report the first GF translocon mutants and unveil CRL as a novel functional partner of TIC236 for PDM import.

Chloroplast import of an intermembrane space protein is facilitated by translocon components Toc75 and Tic236.

Chloroplasts are divided into six subcompartments: the outer membrane, intermembrane space, and inner membrane of the envelope, the stroma, the thylakoid membrane, and the thylakoid lumen. Compared with our knowledge of protein import into other subcompartments, extremely little is known about how proteins are imported into the intermembrane space of the envelope. Tic22 was one of the first proteins identified as localizing to the intermembrane space and the only one for which import has been analyzed in some detail. However, conflicting results have been obtained concerning whether the general translocon is used to import Tic22 into the intermembrane space. Taking advantage of available translocon component mutants, we reanalyzed import of Tic22. We reveal reduced in vitro import of Tic22 preprotein (prTic22) into chloroplasts isolated from the Arabidopsis mar1 and tic236 mutants, which are functional knockdown mutants of the outer-membrane channel Toc75 and the intermembrane space linker Tic236, respectively. Import competition experiments also showed that prTic22 import was reduced by excess amounts of a stroma-targeted preprotein. Our results indicate that prTic22 uses at least part of the general translocon for import into the intermembrane space.

Increased ratio of galactolipid MGDG : DGDG induces jasmonic acid overproduction and changes chloroplast shape.

Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG-synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown. We introduced an inducible microRNA (ami-MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA-biosynthesis and JA-responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated. Expression of JA-biosynthesis and JA-responsive genes were reduced in amiR-MGD1-transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued. Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.

Tissue-Specific Regulation of Plastid Protein Import via Transit-Peptide Motifs.

Plastids differentiate into various functional types (chloroplasts, leucoplasts, chromoplasts, etc.) that have distinct proteomes depending on the specific tissue. Most plastid proteins are encoded by the nuclear genome, synthesized as higher molecular mass preproteins with an N-terminal transit peptide, and then posttranslationally imported from the cytosol. Evidence for tissue-specific regulation of import into plastids, and subsequent modulation of plastid proteomes, has been lacking. We quantified protein import into isolated pea (Pisum sativum) leaf chloroplasts and root leucoplasts and identified two transit-peptide motifs that specifically enhance preprotein import into root leucoplasts. Using a plastid preprotein expressed in both leaves and roots of stable transgenic plants, we showed that losing one of the leucoplast motifs interfered with its function in root leucoplasts but had no effect on its function in leaf chloroplasts. We assembled a list of all Arabidopsis (Arabid opsis thaliana) plastid preproteins encoded by recently duplicated genes and show that, within a duplicated preprotein pair, the isoform bearing the leucoplast motif usually has greater root protein abundance. Our findings represent a clear demonstration of tissue-specific regulation of organelle protein import and suggest that it operates by selective evolutionary retention of transit-peptide motifs, which enhances import into specific plastid types.

TIC236 links the outer and inner membrane translocons of the chloroplast.

The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC-TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that-unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation3,4-a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB-BamA protein-secretion system.

Developmental regulation of protein import into plastids.

The plastid proteome changes according to developmental stages. Accruing evidence shows that, in addition to transcriptional and translational controls, preprotein import into plastids is also part of the process regulating plastid proteomes. Different preproteins have distinct preferences for plastids of different tissues. Preproteins are also divided into at least three age-selective groups based on their import preference for chloroplasts of different ages. Both tissue and age selectivity are determined by the transit peptide of each preprotein, and a transit-peptide motif for older-chloroplast preference has been identified. Future challenges lie in identifying other motifs for tissue and age selectivity, as well as in identifying the receptor components that decipher these motifs. Developmental regulation also suggests that caution should be exercised when comparing protein import data generated with plastids isolated from different tissues or with chloroplasts isolated from plants of different ages.

Chloroplast Galactolipids: The Link Between Photosynthesis, Chloroplast Shape, Jasmonates, Phosphate Starvation and Freezing Tolerance.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) together constitute approximately 80% of chloroplast lipids. Apart from facilitating the photosynthesis light reaction in the thylakoid membrane, these two lipids are important for maintaining chloroplast morphology and for plant survival under abiotic stresses such as phosphate starvation and freezing. Recently it was shown that severe growth retardation phenotypes of the DGDG-deficient mutant dgd1 were due to jasmonate overproduction, linking MGDG and DGDG homeostasis with phytohormone production and suggesting MGDG as a major substrate for jasmonate biosynthesis. Induction of jasmonate synthesis and jasmonic acid (JA) signaling was also observed under conditions of phosphate starvation. We hypothesize that when DGDG is recruited to substitute for phospholipids in extraplastidic membranes during phosphate deficiency, the altered MGDG to DGDG ratio in the chloroplast envelope triggers the conversion of galactolipids into jasmonates. The conversion may contribute to rebalancing the MGDG to DGDG ratio rapidly to maintain chloroplast shape, and jasmonate production can reduce the growth rate and enhance predator deterrence. We also hypothesize that other conditions, such as suppression of dgd1 phenotypes by trigalactosyldiacylglycerol (tgd) mutations, may all be linked to altered jasmonate production, indicating that caution should be exercised when interpreting phenotypes caused by conditions that may alter the MGDG to DGDG ratio at the chloroplast envelope.

Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC-TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC-TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC-TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.

Chloroplast Preproteins Bind to the Dimer Interface of the Toc159 Receptor during Import.

Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import.

Polypeptide Transport-Associated Domains of the Toc75 Channel Protein Are Located in the Intermembrane Space of Chloroplasts.

Toc75 is the channel for protein translocation across the chloroplast outer envelope membrane. Toc75 belongs to the Omp85 protein family and consists of three N-terminal polypeptide transport-associated (POTRA) domains that are essential for the functions of Toc75, followed by a membrane-spanning ß-barrel domain. In bacteria, POTRA domains of Omp85 family members are located in the periplasm, where they interact with other partner proteins to accomplish protein secretion and outer membrane protein assembly. However, the orientation and therefore the molecular function of chloroplast Toc75 POTRA domains remain a matter of debate. We investigated the topology of Toc75 using bimolecular fluorescence complementation and immunogold electron microscopy. Bimolecular fluorescence complementation analyses showed that in stably transformed plants, Toc75 N terminus is located on the intermembrane space side, not the cytosolic side, of the outer membrane. Immunogold labeling of endogenous Toc75 POTRA domains in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana) confirmed that POTRA domains are located in the intermembrane space of the chloroplast envelope.

Reduced Biosynthesis of Digalactosyldiacylglycerol, a Major Chloroplast Membrane Lipid, Leads to Oxylipin Overproduction and Phloem Cap Lignification in Arabidopsis.

DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA.

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process.

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.

Protein import into isolated pea root leucoplasts.

Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that prefer Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.

Evolution of chloroplast J proteins.

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.

Transit peptide design and plastid import regulation.

Import of most nuclear encoded proteins into plastids is directed by an N-terminal transit peptide. Early studies suggested that transit peptides are interchangeable between precursor proteins. However, emerging evidence shows that different transit peptides contain different motifs specifying their preference for certain plastid types or ages. In this opinion article, we propose a 'multi-selection and multi-order' (M&M) model for transit peptide design, describing each transit peptide as an assembly of motifs for interacting with selected translocon components. These interactions determine the preference of the precursor for a particular plastid type or age. Furthermore, the order of the motifs varies among transit peptides, explaining why no consensus sequences have been identified through linear sequence comparison of all transit peptides as one group.

Structural characterizations of the chloroplast translocon protein Tic110.

Tic110 is a major component of the chloroplast protein import translocon. Two functions with mutually exclusive structures have been proposed for Tic110: a protein-conducting channel with six transmembrane domains and a scaffold with two N-terminal transmembrane domains followed by a large soluble domain for binding transit peptides and other stromal translocon components. To investigate the structure of Tic110, Tic110 from Cyanidioschyzon merolae (CmTic110) was characterized. We constructed three fragments, CmTic110A , CmTic110B and CmTic110C , with increasing N-terminal truncations, to perform small-angle X-ray scattering (SAXS) and X-ray crystallography analyses and Dali structural comparison. Here we report the molecular envelope of CmTic110B and CmTic110C determined by SAXS, and the crystal structure of CmTic110C at 4.2 Å. Our data indicate that the C-terminal half of CmTic110 possesses a rod-shaped helix-repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT-repeat motif that functions as scaffolds for protein-protein interactions.

Differential age-dependent import regulation by signal peptides.

Gene-specific, age-dependent regulations are common at the transcriptional and translational levels, while protein transport into organelles is generally thought to be constitutive. Here we report a new level of differential age-dependent regulation and show that chloroplast proteins are divided into three age-selective groups: group I proteins have a higher import efficiency into younger chloroplasts, import of group II proteins is nearly independent of chloroplast age, and group III proteins are preferentially imported into older chloroplasts. The age-selective signal is located within the transit peptide of each protein. A group III protein with its transit peptide replaced by a group I transit peptide failed to complement its own mutation. Two consecutive positive charges define the necessary motif in group III signals for older chloroplast preference. We further show that different members of a gene family often belong to different age-selective groups because of sequence differences in their transit peptides. These results indicate that organelle-targeting signal peptides are part of cells' differential age-dependent regulation networks. The sequence diversity of some organelle-targeting peptides is not a result of the lack of selection pressure but has evolved to mediate regulation.