John E. Froehlich

ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery

Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5. The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A GFP–ARC5 fusion protein localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes.

A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

A chloroplastic outer envelope membrane protein of 75 kDa (OEP75) was identified previously as a component of the protein import machinery. Here we provide additional evidence that OEP75 is a component of protein import, present the isolation of a cDNA clone encoding this protein, briefly describe its developmental expression and tissue specificity, and characterize its insertion into the outer envelope membrane. OEP75 was synthesized as a higher molecular weight precursor (prOEP75) which bound to isolated chloroplasts in an in vitro import assay and subsequently was processed to the mature form (mOEP75). During this import assay, two proteins intermediate in size between prOEP75 and mOEP75 were detected. One of these intermediates was also detected in chloroplast envelopes isolated from young pea leaves. Binding and processing of prOEP75 required ATP and one or more surface‐exposed proteinaceous components, and was competed by prSSU, a stromal‐targeted protein. We propose that the N‐terminus of the prOEP75 transit peptide acts as a stromal‐targeting domain and a central, hydrophobic region of this transit peptide acts as a stop‐transfer domain. A complex route of insertion and processing of prOEP75 may exist to ensure high fidelity targeting of this import component.

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2.

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6–green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site–determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.

A permease‐like protein involved in ER to thylakoid lipid transfer in Arabidopsis

In eukaryotes, enzymes of different subcellular compartments participate in the assembly of membrane lipids. As a consequence, interorganelle lipid transfer is extensive in growing cells. A prominent example is the transfer of membrane lipid precursors between the endoplasmic reticulum (ER) and the photosynthetic thylakoid membranes in plants. Mono‐ and digalactolipids are typical photosynthetic membrane lipids. In Arabidopsis, they are derived from one of two pathways, either synthesized de novo in the plastid, or precursors are imported from the ER, giving rise to distinct molecular species. Employing a high‐throughput robotic screening procedure generating arrays of spot chromatograms, mutants of Arabidopsis were isolated, which accumulated unusual trigalactolipids. In one allelic mutant subclass, trigalactosyldiacylglycerol1, the primary defect caused a disruption in the biosynthesis of ER‐derived thylakoid lipids. Secondarily, a processive galactosyltransferase was activated, leading to the accumulation of oligogalactolipids. Mutations in a permease‐like protein of the outer chloroplastic envelope are responsible for the primary biochemical defect. It is proposed that this protein is part of a lipid transfer complex.

The Hydrophilic Domain of Tic110, an Inner Envelope Membrane Component of the Chloroplastic Protein Translocation Apparatus, Faces the Stromal Compartment

It has previously been found that Tic110, an integral protein of the chloroplast inner envelope membrane, is a component of the chloroplastic protein import apparatus. However, conflicting reports exist concerning the topology of this protein within the inner envelope membrane. In this report, we provide evidence that indicates that the large (>90-kDa) hydrophilic domain of Tic110 is localized within the chloroplast stroma. Trypsin, a protease that cannot penetrate the permeability barrier of the inner envelope membrane, degrades neither Tic110 nor other proteins exposed to the stromal compartment but is able to digest proteins exposed to the intermembrane space between the two envelope membranes. Previous reports indicating that trypsin is able to degrade Tic110 were influenced by incomplete quenching of protease activity. When trypsin is not sufficiently quenched, it is able to digest Tic110, but only after chloroplasts have been ruptured. It is therefore necessary to employ adequate quenching protocols, such as the one reported here, whenever trypsin is utilized as an analytical tool. Based on a stromal localization for the majority of Tic110, we propose that this protein may be involved in the recruitment of stromal factors, possibly molecular chaperones, to the translocation apparatus during protein import.

Mutation of the TGD1 Chloroplast Envelope Protein Affects Phosphatidate Metabolism in Arabidopsis

Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.

A Heteromeric Plastidic Pyruvate Kinase Complex Involved in Seed Oil Biosynthesis in Arabidopsis

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in developing seeds of Arabidopsis thaliana and oil crops. Compartmentation of primary metabolism in developing embryos poses a significant challenge for testing this hypothesis and for the engineering of seed biomass production. It also raises the question whether there is a preferred route of carbon from imported photosynthate to seed oil in the embryo. Plastidic pyruvate kinase catalyzes a highly regulated, ATP-producing reaction of glycolysis. The Arabidopsis genome encodes 14 putative isoforms of pyruvate kinases. Three genes encode subunits α, β1, and β2 of plastidic pyruvate kinase. The plastid enzyme prevalent in developing seeds likely has a subunit composition of 4α4β1, is most active at pH 8.0, and is inhibited by Glu. Disruption of the gene encoding the β1 subunit causes a reduction in plastidic pyruvate kinase activity and 60% reduction in seed oil content. The seed oil phenotype is fully restored by expression of the β1 subunit–encoding cDNA and partially by the β2 subunit–encoding cDNA. Therefore, the identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into oil, suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty acids.

Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato Species Lycopersicon hirsutum Identify a Key Enzyme in the Biosynthesis of Methylketones

Medium-length methylketones (C7-C15) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C12, C14, and C16 β-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C11, C13, and C15 methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site.

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of division.

Arabidopsis ARC6 Coordinates the Division Machineries of the Inner and Outer Chloroplast Membranes through Interaction with PDV2 in the Intermembrane Space

Chloroplasts arose from a free-living cyanobacterial endosymbiont and divide by binary fission. Division involves the assembly and constriction of the endosymbiont-derived, tubulin-like FtsZ ring on the stromal surface of the inner envelope membrane and the host-derived, dynamin-like ARC5 ring on the cytosolic surface of the outer envelope membrane. Despite the identification of many proteins required for plastid division, the factors coordinating the internal and external division machineries are unknown. Here, we provide evidence that this coordination is mediated in Arabidopsis thaliana by an interaction between ARC6, an FtsZ assembly factor spanning the inner envelope membrane, and PDV2, an ARC5 recruitment factor spanning the outer envelope membrane. ARC6 and PDV2 interact via their C-terminal domains in the intermembrane space, consistent with their in vivo topologies. ARC6 acts upstream of PDV2 to localize PDV2 (and hence ARC5) to the division site. We present a model whereby ARC6 relays information on stromal FtsZ ring positioning through PDV2 to the chloroplast surface to specify the site of ARC5 recruitment. Because orthologs of ARC6 occur in land plants, green algae, and cyanobacteria but PDV2 occurs only in land plants, the connection between ARC6 and PDV2 represents the evolution of a plant-specific adaptation to coordinate the assembly and activity of the endosymbiont- and host-derived plastid division components.