A Small ATPase Protein of Arabidopsis, TGD3, Involved in Chloroplast Lipid Import

Abstract

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5′ of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram-bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis. Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5′ of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram-bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis. Interorganellar non-vesicular lipid trafficking in plant cells is required for the biogenesis of the characteristic plant cell organelle, the chloroplast (1Benning C. Xu C. Awai K. Curr. Opin. Plant Biol. 2006; 9: 241-247Crossref PubMed Scopus (63) Google Scholar). The predominant polar lipids found in the photosynthetic membranes (thylakoids) of chloroplasts are the galactoglycerolipids monogalactosyldiacylglycerol (MGDG) 3The abbreviations used are: Col-2, Columbia-2; NAP11, non-intrinsic ABC protein 11; TGDG, trigalactosyldiacylglycerol; MBP, maltose-binding protein; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; ER, endoplasmic reticulum; PtdOH, phosphatidic acid; ABC, ATP binding cassette; MS, mass spectrometry. and digalactosyldiacylglycerol (DGDG) (2Dörmann P. Benning C. Trends Plant Sci. 2002; 7: 112-118Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Their final assembly from diacylglycerol and UDP-galactose requires two lipid galactosyltransferases, which in the model plant Arabidopsis are designated MGD1 and DGD1 (3Benning C. Ohta H. J. Biol. Chem. 2005; 280: 2397-2400Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Of these two enzymes MGD1 is associated with the outside of the inner chloroplast envelope membrane (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar) and is responsible for the bulk of MGDG formation (5Jarvis P. Dörmann P. Peto C.A. Lutes J. Benning C. Chory J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8175-8179Crossref PubMed Scopus (210) Google Scholar), whereas DGD1 is located on the outside of the outer chloroplast envelope membrane (6Froehlich J.E. Benning C. Dörmann P. J. Biol. Chem. 2001; 276: 31806-31812Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and is required for the bulk of DGDG biosynthesis (7Dörmann P. Balbo I. Benning C. Science. 1999; 284: 2181-2184Crossref PubMed Scopus (163) Google Scholar). The diacylglycerol moiety of the galactoglycerolipids is either synthesized de novo in the plastid or it is assembled at the endoplasmic reticulum (ER) and needs to be imported into the plastid (8Roughan P.G. Slack C.R. Annu. Rev. Plant Physiol. 1982; 33: 97-132Crossref Google Scholar). Understanding this complexity of subcellular organization of glycerolipid biosynthesis in plants and the underlying lipid trafficking phenomena still poses some of the biggest challenges of modern plant biochemistry. The recent isolation and characterization of genetic suppressors of the DGDG-deficient dgd1 mutant of Arabidopsis has provided the first components, TGD1 and TGD2, of a potential lipid transport complex associated with the inner chloroplast envelope membrane in Arabidopsis (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar, 9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar, 10Xu C. Fan J. Riekhof W. Froehlich J.E. Benning C. EMBO J. 2003; 22: 2370-2379Crossref PubMed Scopus (166) Google Scholar). The TGD1 protein is similar to the permease component of bacterial ATP binding cassette (ABC) transporters, and the Arabidopsis tgd1 mutant shows a complex lipid phenotype; that is, the accumulation of oligogalactolipids and triacylglycerols, a disruption of the assembly of galactolipids from ER-derived precursors, and the elevation of phosphatidic acid (PtdOH) levels (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar, 10Xu C. Fan J. Riekhof W. Froehlich J.E. Benning C. EMBO J. 2003; 22: 2370-2379Crossref PubMed Scopus (166) Google Scholar). The name of the mutants, tgd (trigalactosyldiacylglycerol), refers to the characteristic accumulation of oligogalactolipids. Presumably, this is a secondary phenotype due to the activation of a processive galactosyl transferase of unknown function, for which the gene has not yet been identified. The accumulation of PtdOH and reduced incorporation of labeled PtdOH into galactoglycerolipids by isolated tgd1 chloroplasts led to the suggestion that TGD1 is involved in the transport of PtdOH through the inner chloroplast envelope membrane (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar). The TGD2 protein is similar to membrane-tethered substrate-binding proteins associated with bacterial ABC transporters (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar). Although not as extensively characterized as the tgd1 mutant, the Arabidopsis tgd1 and tgd2 mutants have a nearly identical lipid phenotype in all aspects tested. Moreover, the recombinant TGD2 protein lacking the single membrane-spanning domain specifically binds PtdOH (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar). The nearly identical phenotypes of the two mutants and the organization of bacterial orthologs of TGD1 and TGD2 in operons (Fig. 1A) strongly suggest that both are involved in a common biochemical or cell biological process. Moreover, the specific binding of PtdOH to TGD2 corroborates the hypothesis that the two proteins are components of a PtdOH transporter at the inner chloroplast envelope. Unlike other eukaryotic ABC transporters or the MsbA lipid transporter from Escherichia coli (11Doerrler W.T. Mol. Microbiol. 2006; 60: 542-552Crossref PubMed Scopus (81) Google Scholar), the TGD1 protein lacks an ATP binding domain. However, the Arabidopsis genome contains 26 genes encoding putative small ATPases with either one or two nucleotide binding domains (12Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). We hypothesized that one of these could encode a small ATPase potentially associated with the TGD1 protein. Here we describe the identification and biochemical characterization of TGD3, the small ATPase protein proposed to be associated with TGD1 and TGD2. Plant Materials and Growth Conditions—Arabidopsis thaliana wild type and tgd1 and tgd2 mutant plants (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar, 10Xu C. Fan J. Riekhof W. Froehlich J.E. Benning C. EMBO J. 2003; 22: 2370-2379Crossref PubMed Scopus (166) Google Scholar) were of the ecotype Columbia-2 (Col-2). The following T-DNA insertion lines were made available by the Salk Institute Genome Analysis Laboratory (SIGnAL) and were obtained from the Arabidopsis Biological Resource Center at Ohio State University: SALK_027270 At4g33460, SALK_129048 At5g60790, SALK_029696 At5g09930, SALK_113472 At5g64840, SALK_116866 At5g14100, SALK_055996 At1g32500, and SALK_040335 At1g65410. Homozygous mutant lines were screened and used for all experiments. Seeds were surface-sterilized and grown on 0.8% (w/v) agar-solidified Murashige-Skoog medium (13Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53969) Google Scholar) supplemented with 1% (w/v) sucrose for 5 days. Wild-type and mutant seedlings of similar developmental stage were then transferred to a fresh Murashige-Skoog agar plate with 1% sucrose and grown for three additional weeks before lipid analysis. Genotyping and Complementation—The TGD3 open reading frame was isolated by reverse transcriptase-PCR (standard conditions) from mRNA preparations using RNeasy and Omniscript kits (Qiagen, Valencia, CA) with the primers 5′-ACGGTACCATGCTTTCGTTATCATGCTC-3′ and 5′-CTGGTACCCTAGTATCTGATTGGTCCAT-3′. The PCR products were ligated into pGEM-Teasy (Promega, Madison, WI) and sequenced. The resulting plasmids were digested with KpnI and inserted into pCAMBIA1300 (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar) followed by transformation into Agrobacterium. Plants were transformed by the floraldip method Plant J. PubMed Google Scholar) and screened for to on agar-solidified Murashige-Skoog an of TGD3 levels by reverse was isolated from and tgd3 mutant and reverse was using primers The primers were used for the PCR The of was for The following primers were and the analysis, was from and the following primers were T-DNA The PCR were followed by at for for and for followed by a final of 5 at Lipid were from and tgd3 mutant seedlings grown on Murashige-Skoog medium with 1% acid were as P. S. Balbo I. Benning C. Plant Cell. 7: PubMed Scopus Google Scholar) and by to A. Xu Benning C. PubMed Scopus Google Scholar). Polar lipids were on P. S. Balbo I. Benning C. Plant Cell. 7: PubMed Scopus Google Scholar) with lipids were on with acid were by to or with to C. PubMed Scopus Google Scholar). The acid at the of lipids were using to and Scopus Google Scholar) with as in C. J. Plant Physiol. PubMed Scopus Google Scholar). acid were from and the acid were by were characterized using on a mass with and were using and in both and similar to T. K. T. R. 2005; PubMed Scopus Google Scholar) in of mass were in at and on one to with and The for of characteristic that were using a and a on and the of a protein the coding of was inserted into the KpnI of (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar) using primers 5′-ACGGTACCATGCTTTCGTTATCATGCTC-3′ and of the in and of the resulting was as (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar). the in using chloroplasts the and were used as for the of the MGD1 protein (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar). and coding for TGD3 to a protein missing as was As the pGEM-Teasy was used in with primers and The was inserted into pGEM-Teasy and sequenced. This as for by using the primers and the from The and reverse primers for are and The are for the with and either the wild type or the mutant was inserted of the coli coding into the to the maltose-binding protein proteins or recombinant protein a was grown at with of an The protein was with at an of and at for The cells were then by and by in the of 1 was at for and the was an proteins were to was to PubMed Scopus Google Scholar) using as a was by were at for a weeks of for ATPase protein was for ATPase activity at a protein of in The protein was in to a final of the of ATP in the from to from a made in the were a final ATP of to 1 ATPase were at for the and by the of was by a as by S. A. R. S. PubMed Scopus Google Scholar) using as the the of of a of acid in 1 and were and the were at for 5 This was followed by the of of a of and of at was at The was in the of of The activity was for of from ATP and for by obtained in the of protein and A was used to for ATPase activity from The were by to the using of TGD3 by and genes involved in the biochemical are in This has been in genome or the identification of genes A. R. Curr. Opin. Chem. Biol. 2003; 7: PubMed Scopus Google Scholar). the of TGD1 and TGD2 encoding in bacterial operons (Fig. 1A) to putative ABC proteins that these are the ABC components of the respective transport Based on it that Arabidopsis has an ABC protein associated with the extensive and of transport ATPases and non-intrinsic ABC proteins potentially associated with membrane transporters in Arabidopsis has been (12Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) 26 nucleotide binding domain TGD1 and TGD2 are associated with the inner chloroplast which is to the bacterial cell we that the respective ABC protein was localized on the inside of the inner chloroplast membrane an chloroplast of the ABC proteins were predicted to be to the plastid using H. Sci. 1999; PubMed Scopus Google At1g32500, At4g33460, At5g09930, At5g14100, At5g60790, of these T-DNA lines H. P. J. P. R. C. C. A. H. H. C. R. I. P. T. A. Science. 2003; PubMed Scopus Google Scholar) were available at the and were and for The mutant lines were for the of which is for mutants with TGD1 or TGD2 one carrying a T-DNA 5′ of the of Arabidopsis gene (Fig. accumulation of the and This mutant was as the of the respective mRNA by reverse transcriptase-PCR was reduced in the T-DNA (Fig. A disrupted was not available phenotype of the tgd3 mutant with the tgd2 mutant, and wild lipids were from seedlings grown on Murashige-Skoog agar 1% sucrose and by of lipids. were by to of polar lipids. were by that the mutant phenotype was not due to secondary a was in the T-DNA disrupted in At1g65410. Two T-DNA lines lacking the accumulation of the are in and The of the phenotype in the T-DNA lines by the that the accumulation of oligogalactolipids in the T-DNA lines was due to the disruption of the the SALK_040335 be to as tgd3 mutant the characterized mutant The protein was designated ABC (12Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) and be to as trigalactosyldiacylglycerol with the acid of TGD3 with proteins by bacterial using J. W. PubMed Scopus Google Scholar), ABC open reading to putative provided some of the with putative TGD3 from (Fig. and the coli The tgd3 a Lipid with tgd is to that all three tgd mutant and are to of the as for TGD1 (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar) mutants to analysis. the TGD3 protein is part of a lipid transport complex with TGD1 and TGD2, mutant lipid phenotypes for all three be lipid from the and the tgd3 lines were by of (Fig. and polar lipids (Fig. The tgd3 mutant a lipid with (Fig. in the tgd1 mutant (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar) and to accumulate in tgd2 mutant (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar). polar lipid of the tgd3 mutant a lipid with which is for the of (Fig. This lipid with trigalactosyldiacylglycerol in tgd1 and of the of and lipids was provided by of of wild type and tgd3 mutants (Fig. in a at for the tgd3 mutant to for which was the lipid in the The this in the of a at to the acid were characterized using at to to of triacylglycerols with and a of from to (Fig. for in the of in the tgd3 mutant the wild type (Fig. and for both and lipid were with of acid by to The lipid of the tgd3 mutant from that of the wild type as in the of the chloroplast lipids MGDG and DGDG were whereas the of galactolipids from the (8Roughan P.G. Slack C.R. Annu. Rev. Plant Physiol. 1982; 33: 97-132Crossref Google Scholar), was Analysis of the acid of MGDG and DGDG distinct in the acid in tgd3 as with the wild type (Fig. and were and acid was Moreover, the tgd3 acid were similar to for the tgd1 and tgd2 mutants (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar). the tgd mutants in the have an acid (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar) in This is for DGDG, which is to a from the Plant Physiol. PubMed Google Scholar). the for the from to in the tgd3 mutant (Fig. This is due to the substrate of the in the plastid and the to at the and at the of the diacylglycerol for lipids. lipids from the in both Plant Physiol. PubMed Google Scholar). using an in in the of MGDG and DGDG of the tgd3 tgd3 has a complex lipid phenotype similar to tgd1 and tgd2 that is with an of the of This in phenotypes that the TGD1, TGD2, and TGD3 proteins are involved in the biochemical process. The TGD3 into the TGD3 has a predicted chloroplast the of the the was with a protein and in The in shows the protein associated with which are by the of TGD3, in labeled TGD3 protein was with isolated Two were used to the a that domains of outer envelope membrane proteins as is K. J. K. Plant Physiol. PubMed Google Scholar), and which the outer envelope membrane not the inner Froehlich J.E. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of this is in The TGD3 was with the of a Moreover, the TGD3 protein was to and was digested by in the of were with of TGD3 into the chloroplast beyond the inner chloroplast envelope membrane. Based on these TGD3 be available for with the complex at the of the inner envelope membrane. TGD3 ATPase TGD3 protein contains characteristic for other small ATPases associated with bacterial membrane transporters 2006; PubMed Scopus Google Scholar, J. Annu. Rev. PubMed Scopus Google Scholar) designated and (Fig. the biochemical of TGD3, an missing the coding for the first the predicted was to the of the coli maltose-binding protein open reading frame and in this was the TGD3 protein. of the recombinant protein designated on an is in The protein was and as a protein. The protein in the was for to maltose-binding protein by or a mutant to an mutant of TGD3 in the as the wild type were be that the not protein or protein the to in TGD3 in bacterial orthologs with a was to the transport J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). as the was for (Fig. the with protein in the (Fig. the of a substrate was for the protein (Fig. on this were for the and for are in the as for ATP by other ABC transporters S. Microbiol. Rev. 22: PubMed Google Scholar). The mutant protein activity for of the (Fig. The maltose-binding protein The identification of TGD1 and TGD2 proteins and the characterization of the respective mutants (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar, 9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar, 10Xu C. Fan J. Riekhof W. Froehlich J.E. Benning C. EMBO J. 2003; 22: 2370-2379Crossref PubMed Scopus (166) Google Scholar) provided suggesting that these two proteins are involved in PtdOH from the outer envelope through the inner envelope membrane of chloroplasts in Arabidopsis. lipid transporters as the characterized MsbA protein from coli W.T. C.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, W.T. C.R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) of a of a with a membrane-spanning permease domain and a ATP binding domain. the of the TGD1 the permease domain and TGD2 is similar to a membrane-tethered substrate binding protein associated with other bacterial ABC The TGD3 protein of Arabidopsis to be the missing ATP binding component that be required for a complex as proposed in lines of are with the current hypothesis for the of the PtdOH transporter of the inner chloroplast envelope membrane and the of the TGD3 protein; and potential orthologs of the three Arabidopsis proteins the TGD3 protein. Their respective genes are in operons with in the biochemical (Fig. Of the 26 genes of Arabidopsis encoding small proteins membrane-spanning domains (12Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), were predicted to be to the Of the genes the tgd3 mutant to the gene encoding TGD3 a phenotype identical to the characterized tgd1 and tgd2 The TGD3 protein was localized to the of chloroplasts and ATPase activity in as a maltose-binding protein the ATPase component of the transport complex and of biochemical transport activity in be However, current have been by the that the of TGD1 and TGD2 proteins in coli has not yet been the TGD3 protein could be to maltose-binding protein. The in lipid transport by membrane are from the that for the MsbA protein of lipid the membrane is for lipid transport by MsbA is on the respective mutant phenotype W.T. C.R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) and the that specific lipids the ATPase activity of MsbA in W.T. C.R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of ATPase activity of TGD3 was not a the MsbA the TGD3 protein lacks the permease domain that with the lipid As for the complex of Arabidopsis, the of MsbA still a mutant of lacking the MsbA of of the outer membrane B. F. J. de H. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). from and the lipid of or lipid these we are the of the complex in the inner chloroplast membrane. the for TGD1 or TGD2 open the for TGD3 TGD1 was in chloroplast (4Xu C. Fan J. Froehlich J.E. Awai K. Benning C. Plant Cell. 2005; 17: 3094-3110Crossref PubMed Scopus (159) Google Scholar, 10Xu C. Fan J. Riekhof W. Froehlich J.E. Benning C. EMBO J. 2003; 22: 2370-2379Crossref PubMed Scopus (166) Google Scholar), and it that at of TGD1 be localized in the inner and outer chloroplast envelope membranes. the TGD1 protein could be present in a the two membranes. TGD2 protein was to the protein protein was not (9Awai K. Xu C. Tamot B. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10817-10822Crossref PubMed Scopus (158) Google Scholar). This could be as the of TGD2 in localized domains the inner and the outer envelope which be to the other the for TGD3 is TGD3 is imported into the chloroplast beyond the inner envelope membrane and in (Fig. The TGD3 could with TGD1 and TGD2 as by the identical phenotypes for the respective mutants and the organization of predicted bacterial orthologs is TGD1 and TGD2 are proteins of the inner chloroplast envelope membrane as in TGD1 and TGD2 a of the three proteins in the inner chloroplast envelope has in using available in either the isolation of the complex or from recombinant both of which have not yet been However, current suggest that TGD3 is the missing ATPase of this a to the of a transporter of the TGD1 permease and the TGD3 protein. We Froehlich from the State of Plant Laboratory for on the chloroplast experiments. we R. from the State of and for on protein and with