Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Riekhof, W. R. Articles by Benning, C. Search for Related Content PubMed PubMed Citation Articles by Riekhof, W. R. Articles by Benning, C.

 Previous Article  |  Next Article 

Eukaryotic Cell, February 2005, p. 242-252, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.242-252.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1_Cr

Wayne R. Riekhof,^1,2 Barbara B. Sears,³ and Christoph Benning^1*

Department of Biochemistry and Molecular Biology,¹ Department of Plant Biology,³ Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, Michigan²

Received 30 July 2004/ Accepted 8 October 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1_Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1_Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1_Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaA_Rs and BtaB_Rs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1_Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaA_Rs and BtaB_Rs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Knowledge of genes and proteins involved in lipid metabolism in plants has greatly increased over the past decade. Many genes encoding enzymes of glycerolipid biosynthesis and fatty acid desaturation have been identified by genetic and biochemical means (10, 16, 33), and even lipid trafficking phenomena are beginning to be unraveled by genetic approaches (57). In addition, the availability of the Arabidopsis genome sequence (51) has facilitated the annotation of many uncharacterized gene products which are likely to have activities in lipid biosynthesis, trafficking, and catabolism (4). Although Arabidopsis is undoubtedly the most widely used plant system currently under investigation, the eukaryotic green alga Chlamydomonas reinhardtii is a well-established model for many processes, such as photosynthesis (31), phototaxis and flagellar function (47), posttranscriptional gene silencing (56), and nutrient acquisition (8). Recent large-scale expressed sequence tag (EST) projects and sequencing of the C. reinhardtii genome (21, 46) and the development of insertional mutagenesis (50), RNA interference methods (17, 48), and a molecular map (25) make Chlamydomonas an attractive genetic model. These attributes make Chlamydomonas also an ideal model organism with which to study the biosynthesis and physiological functions of nonphosphorous lipids, such as the plastidic galactolipids and sulfolipids and the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) (18, 29, 39), as well as phospholipids, such as phosphatidylethanolamine (PtdEtn) (58) and phosphatidylglycerol (PtdGro) (11, 34, 45).

We have begun to explore Chlamydomonas as a model for plant lipid metabolism by using a high-throughput robotic screening method to find mutants with defects in lipid metabolism (35). The initial goal of the study reported here was to identify and annotate genes involved in acyl-lipid metabolism in the Chlamydomonas version 2.0 draft genome sequence available at http://genome.jgi-psf.org/chlamy/. Enzymes of fatty acid biosynthesis, glycerolipid synthesis, and fatty acid desaturation were identified and organized so as to reconstruct lipid biosynthesis pathways and posit their subcellular localizations. As a specific example, this study allowed us to identify and characterize the enzyme responsible for the synthesis of the betaine lipid DGTS, which replaces the phospholipid phosphatidylcholine (PtdCho) in extraplastidic membranes of Chlamydomonas. Due to its structural similarity to PtdCho and the lack of PtdCho in Chlamydomonas, DGTS is presumed to take on the functions of PtdCho as the major component of the extraplastidic membranes of this organism (29, 40, 42).

Previous work elucidated the pathway for DGTS biosynthesis in Rhodobacter sphaeroides (23), and genetic analysis with this bacterium identified two gene products, BtaA and BtaB, which are required for phosphate stress-induced DGTS production in R. sphaeroides (27). The btaA gene was proposed to encode an S-adenosylmethionine (AdoMet):diacylglycerol (DAG) 3-amino-3-carboxypropyltransferase giving rise to the intermediate diacylglycerolhomoserine (DGHS), and the btaB gene was proposed to encode an AdoMet-dependent N-methylase converting DGHS to DGTS. Here we describe the discovery of the C. reinhardtii BTA1 (BTA1_Cr) gene, encoding a Chlamydomonas betaine lipid synthase that was shown to encompass both activities in a single polypeptide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Annotation of C. reinhardtii genes involved in lipid metabolism. Version 2.0 of the C. reinhardtii whole-genome shotgun sequence assembly is available at http://genome.jgi-psf.org/chlamy. Genes encoding enzymes and other components of lipid metabolism (e.g., acyl carrier proteins [ACPs]) were manually annotated based on key word searches executed against the automatically annotated gene loci (21), followed by verification of similarity by BLASTP searches (1) and CLUSTALW multiple-sequence alignments (52) with verified protein sequences. In addition, direct BLASTP and TBLASTN searches of the version 2.0 assembly were carried out with known protein sequences from plants, animals, fungi, and bacterial sources to add additional evidence and to identify genes that were incorrectly annotated by the automated system. For the tentative prediction of subcellular localizations, the programs TargetP and ChloroP were used (12, 13). When the N terminus was not available due to sequence gaps and/or ambiguity in the prediction of the first exon, tentative localizations were predicted on the basis of clustering with isoforms from other species for which subcellular localizations are known.

Materials. Restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, and the Klenow fragment were obtained from New England Biolabs (Beverly, Mass.). SuperScript II RNase H reverse transcriptase was obtained from Invitrogen (Carlsbad, Calif.). Taq and Pfu DNA polymerases were obtained from Stratagene (La Jolla, Calif.). All other chemicals and solvents were of reagent grade and were obtained from Sigma Chemical Co. (St. Loius, Mo.), EM Science (Gibbstown, N.J.), or J. T. Baker Chemical Co. (Phillipsburg, N.J.).

Cloning and expression of BtaA_Rs, BTA1_Cr, and derivatives of BTA1_Cr in Escherichia coli. A summary of all strains and plasmids used in this study is shown in Table 1. The coding region of btaA_Rs was PCR amplified from plasmid pRKL323 (27) for expression in pACYC-31 (9) with the following primers (restriction sites are underlined): btaA forward (SphI), 5'-ACATGCATGCAGTGACGCAGTTCGCCCTC-3', and btaA reverse (KpnI), 5'-CGGGGTACCAGGACGATCCGCTCGAACCG-3'. PCR was carried out with Taq DNA polymerase according to the manufacturer's specifications, except that 10% (vol/vol) dimethyl sulfoxide was added to each reaction mixture to overcome difficulties in PCR due to the high G+C content of R. sphaeroides DNA. The PCR product was cloned into the EcoRI site of pBluescript SK(+), sequenced at the Michigan State University (MSU) Genomics Technology Support Facility, and subcloned into pACYC-31 by using the restriction sites underlined in the PCR primer sequences shown above. The resulting construct was designated pBtaA-LC (btaA in pACYC-31).

View this table: [in this window] [in a new window]

TABLE 1. Strains and plasmids used in this work

C. reinhardtii strain CC125 was grown to mid-log phase in 50 ml of TAP medium (22) and harvested by centrifugation. RNA was purified with Trizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (1.0 µg) was reverse transcribed with SuperScript II RNase H reverse transcriptase. The coding region of BTA1_Cr was amplified from an aliquot of cDNA with Pfu DNA polymerase and the following primers (the native start codon of BTA1_Cr in the forward primer is shown in bold type, and BamHI and KpnI sites are underlined in the forward and in the reverse primers, respectively): CrBTA1 forward, 5'-CAGGATCCAATGGGGTCGGGTCGT-3', and CrBTA1 reverse, 5'-CAGGTACCGCCGCCAGCTGCTTA-3'. The product was cloned in frame with the N-terminal His₆ tag of pQE-31, giving rise to pBTA1.

Site-directed mutagenesis of pBTA1 was carried out with a Quikchange II XL kit (Stratagene) according to the manufacturer's instructions. For the V340A-D341A mutant version of pBTA1 (pBTA1-µA), the mutagenic primers were µA-fw (5'-GCCAGGTGGTGTCGGCGGCATGCAACCCCGCGCAG-3') and its reverse complement µA-rev. For the V121A-D122A mutant (pBTA1-µB), the mutagenic primers were µB-fw (5'-CAAGTCCATCTACGTGGCCGCCCTGTGCCACTCGCTG-3') and its reverse complement µB-rev. Primers were designed such that in addition to the missense mutations for changing codons, a diagnostic restriction site was created or eliminated (e.g., "µB" destroys a SalI site and "µA" introduces an SphI site) in order to easily identify strains carrying mutated plasmids.

pBluescript and pQE-31 derivatives were propagated in Luria-Bertani (LB) medium containing ampicillin at 100 µg/ml, and pACYC-31 derivatives were propagated in LB medium containing chloramphenicol at 25 µg/ml. Strains containing compatible combinations of plasmids were constructed by cotransformation of heat shock-competent cells of TOP10F' (Invitrogen) with 100 ng of each plasmid followed by selection on LB agar containing both ampicillin and chloramphenicol.

In vivo assays of DGHS and DGTS synthesis in E. coli. E. coli TOP10F' cells harboring compatible combinations of plasmids were grown as 2-ml overnight cultures, and 0.1-ml samples of these cultures were used to inoculate 10 ml of LB medium containing appropriate antibiotics. After growth to an optical density at 600 nm of 0.6, the cultures were harvested by centrifugation and dispersed into 10 ml of M9 minimal medium (37) containing 0.2 mM isopropyl-ß-D-thiogalactoside (IPTG) and 0.5 µCi of [1-¹⁴C]methionine (American Radiolabeled Chemicals, St. Louis, Mo.). Cultures were incubated for 3 h and harvested by centrifugation; these steps were followed by extraction with 2 ml of chloroform-methanol (1:1 [vol/vol]) and phase separation by the addition of 0.5 ml of 1 M KCl-0.2 M H₃PO₄. A portion of the organic phase from each extraction was spotted on activated (120°C, 2 h) thin-layer chromatography (TLC) plates (Si 250; Baker), resolved with chloroform-acetone-methanol-acetic acid-water (10:4:2:2:1 [vol/vol]), and subjected to autoradiography.

In vitro BTA1_Cr activity assays. E. coli TOP10F' cells harboring pBTA1 were grown in 250 ml of LB medium-ampicillin at 37°C to an optical density of 0.7 and induced with 0.25 mM IPTG; these steps were followed by an additional 4 h of growth at 28°C. Cells were harvested by centrifugation, and the cell pellet was suspended in 10 ml of cold buffer (50 mM HEPES, 1 mM dithiothreitol, 1 mM EDTA [pH 7.3]). The resuspended cells were sonicated three or four times for 30 s each time with a microprobe tip, and the lysates were centrifuged at 2,000 x g for 10 min to remove unbroken cells and cellular debris. Aliquots (1 ml) of the cell extracts were frozen in liquid N₂ and stored at –80°C prior to use. Activity under these storage conditions did not decrease appreciably for at least 1 month.

Assays for the pH-activity profile were conducted with a 100-µl final volume by combining 48.75 µl of cell extract with 48.75 µl of 100 mM HEPES, Tris-Cl, or morpholineethanesulfonic acid, 1 mM dithiothreitol, and 1 mM EDTA at various initial pHs to give a final pH in the range of 5.5 to 8.6 when mixed with the cell extract (initial pH, 7.3). Reactions were initiated by the addition of 25,000 dpm of [1-¹⁴C]AdoMet (American Radiolabeled Chemicals; 2.5 µl; final concentration of AdoMet, 2.1 µM), incubated for 30 min at 28°C, and terminated by the addition of 400 µl of chloroform-methanol (1:1 [vol/vol]) and 100 µl of 0.9% (wt/vol) NaCl to separate the aqueous and organic phases. Under these conditions, product formation was linear over time up to 1 h at all concentrations of AdoMet tested. The organic phase was transferred to a new tube, dried under a stream of nitrogen, and dissolved in 50 µl of chloroform-methanol (1:1 [vol/vol]). This lipid extract was spotted on silica TLC plates (Baker) and developed with the solvent chloroform-acetone-methanol-acetic acid-water (10:4:2:2:1 [vol/vol]); these steps were followed by quantification of the signal for DGHS and methylated products on a PhosphorImager screen (Molecular Dynamics, Piscataway, N.J.) with the ImageQuant software package. Determination of the K_m was carried out at pH 7.2 with various concentrations of AdoMet by direct fitting of a hyperbola to the experimental data in the Origin software package (OriginLab, Northampton, Mass.).

Structure confirmation for DGTS produced by BTA1_Cr in E. coli. E. coli cells harboring pBTA1 were cultured as described above for the production of crude extracts for enzyme assays. DGTS was isolated from lipid extracts by preparative TLC on ammonium-sulfate-impregnated silica TLC plates with the solvent chloroform-acetone-methanol-acetic acid-water (10:4:2:2:1 [vol/vol]). The DGTS zone was scraped from the plates, eluted with chloroform-methanol (1:1 [vol/vol]), and concentrated under a stream of nitrogen. Fast-atom bombardment-mass spectrometry was done as previously described (5), except that nitrobenzyl alcohol was used as a matrix. ¹H nuclear magnetic resonance (NMR) spectroscopy was conducted by dissolving approximately 200 µg of lipid in CDCl₃, and data were collected as previously described (5).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Annotation of genes involved in acyl-lipid biosynthesis in the Chlamydomonas genome. (i) Overall results. A metabolic reconstruction of glycerolipid metabolism in Chlamydomonas from genomic sequence data was performed in silico. For this purpose, we searched all available genomic sequences of Chlamydomonas, essentially covering the entire genome, for deduced protein sequences with significant similarity to lipid biosynthetic enzymes with experimentally verified activities from plants and other organisms. For building of the pathway models, we also considered analytical data published for the lipid compositions of various membranes in Chlamydomonas and plants in general. Figures 1 and 2, in conjunction with Table 2, show schemes for membrane lipid biosynthesis, starting with fatty acid synthesis in the plastid (Fig. 1, steps 1 to 4 and 16), plastidic glycerolipid biosynthesis (Fig. 1, steps 5 to 15), extraplastidic phospholipid and betaine lipid biosynthesis (Fig. 1, steps 16 to 31), and fatty acid desaturation (Fig. 2, steps 32 to 40). Genes are identified by their version 2.0 gene model transcript number, as well as by three-letter abbreviations based on the tentative assignment of function. These assignments were made in accordance with the recommendations of the Commission on Plant Gene Nomenclature and the Mendel database (28).

View larger version (26K): [in this window] [in a new window]

FIG. 1. Pathways of lipid biosynthesis which are known or hypothesized to occur in Chlamydomonas and their presumed subcellular localizations. Numbers in bold type correspond to the reactions and steps summarized in Table 2. Synthesis of the betaine lipid DGTS (step 23) is highlighted and was characterized in detail. Abbreviations: ACP, acyl carrier protein; AdoMet, S-adenosylmethionine; ASQD, 2'-O-acyl-sulfoquinovosyldiacylglycerol; CDP, cytidine-5'-diphosphate; CoA, coenzyme A; CTP, cytidine-5'-triphosphate; DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; DGTS, diacylglyceryl-N,N,N-trimethylhomoserine; Etn, ethanolamine; FA, fatty acid; G-3-P, glycerol-3-phosphate; Glc, glucose; Glc-1-P, glucose-1-phosphate; Ins, inositol; Ins-3-P, inositol-3-phosphate; Met, methionine; MGDG, mono- galactosyldiacylglycerol; P-Etn, phosphoethanolamine; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGroP, phosphatidylglycerophosphate; PtdIns, phosphatidylinositol; PtdOH, phosphatidic acid; Ser, serine; SQ, sulfoquinovose; SQDG, sulfoquinovosyldiacylglycerol; UDP, uridine-5'-diphosphate.

View larger version (21K): [in this window] [in a new window]

FIG. 2. Pathways of acyl chain desaturation, with numbers in bold type serving to cross reference the specific reactions with the respective enzymes and gene models summarized in Table 2. Fatty acids esterified to the sn1 and sn2 positions of the glycerol moieties of the respective lipids are indicated as "fatty acid at sn1/fatty acid at sn2." Fatty acids are identified as "carbons:double bonds."

View this table: [in this window] [in a new window]

TABLE 2. Assignment of candidate genes encoding enzymes catalyzing the reactions diagrammed in Fig. 1 and 2

(ii) Lipid metabolism in plastids. Genes encoding enzymes of plastidic lipid metabolism are readily identifiable in the genome and appear to be most similar to those of flowering plants. For example, it is clear that all components of the multimeric bacterium-type acetyl coenzyme A (acetyl-CoA) carboxylase (Fig. 1 and Table 2, 1a to 1d) and fatty acid synthase complexes (Table 2, 4a to 4d) are accounted for and that these are most similar to those that have been described for flowering plant plastids. We identified single orthologs for many of the genes encoding enzymes central to fatty acid biosynthesis, e.g., 3-hydroxyacyl-ACP-dehydratase and enoyl-CoA reductase (Table 2, 4c and 4d), components of the type II fatty acid synthase, and the predicted proteins have high and approximately equal probabilities of being targeted to both mitochondria and chloroplasts, as judged by TargetP analysis (12).

The pathway of sulfoquinovosyldiacylglycerol (SQDG) biosynthesis (Fig. 1, steps 11 and 12) has been thoroughly characterized in Arabidopsis and shown to be the function of the products of two genes, namely, SQD1 and SQD2 (14, 59). SQD1 has been characterized in Chlamydomonas by both insertional inactivation (35) and heterologous complementation of a sulfolipid-deficient sqdB cyanobacterial mutant (43), and the SQD1 gene, encoding UDP-sulfoquinovose synthase (Fig. 1, step 11), is highly expressed, as judged by EST abundance. This disproportionately high mRNA abundance might be due to the relatively large amount of SQDG in Chlamydomonas compared to that in Arabidopsis, coupled with the fact that the k_cat for the reaction catalyzed by SQD1 is very low (38), a feature which might necessitate increased abundance of the enzyme. Genes encoding enzymes of galactolipid biosynthesis are also present in the genome, with MGD1 encoding monogalactosyldiacylglycerol synthase (Fig. 1, step 14) and DGD1 encoding digalactosyldiacylglycerol synthase (Fig. 1, step 15).

Several functions in plastidic lipid biosynthesis are still without candidate genes; these include the phosphatidylglycerophosphate phosphatase (Fig. 1, step 9), which is unknown in plants and algae (4), and a putative activity utilizing SQDG and an unidentified acyl donor to form 2'-O-acyl-SQDG (Fig. 1, step 13). 2'-O-Acyl-SQDG has been shown to be a minor component of Chlamydomonas lipids; however, it is apparently absent from flowering plants (35). It should also be noted that, unlike the endoplasmic reticulum (ER) isoforms, the plastidic isoform of phosphatidate phosphatase (Fig. 1, step 10), which is responsible for generating DAG for use in galactolipid and sulfolipid synthesis, is not known yet in flowering plants.

(iii) Extraplastidic lipid metabolism. Membrane lipid metabolism outside the plastid in Chlamydomonas differs from that in flowering plants by both the presence of unique pathways and the apparent absence of certain central pathways and branches of pathways. Most notable is the tentative absence of a gene for phosphoethanolamine methyltransferase, which provides the pathway for the methylation of phosphoethanolamine to form phosphocholine. This activity is responsible for the generation of phosphocholine for conversion into CDP-choline and then PtdCho, and the enzymes from spinach and Arabidopsis have been functionally identified by their ability to rescue a cho2 mutant of Schizosaccharomyces pombe and an opi3 mutant of Saccharomyces cerevisiae, respectively (6, 32). The apparent lack of both a pathway for PtdCho precursor biosynthesis in Chlamydomonas and the PtdEtn methylation pathway, whereby PtdEtn is N trimethylated to form PtdCho, correlates well with the lack of PtdCho in cellular membranes (19).

Phosphatidylserine (PtdSer) is a major precursor of PtdEtn in yeasts, bacteria, and plants through the action of PtdSer decarboxylase (53); however, Chlamydomonas has been reported to lack PtdSer as a component of its membranes (19). This fact is corroborated by our survey of the genome, in that there is an apparent lack of genes encoding PtdSer synthase or phospholipid base exchange enzymes. These data lead us to propose that the biosynthesis of PtdEtn is the function of a single pathway, consisting of serine decarboxylase (36), to generate ethanolamine (Fig. 1, step 25); this step is followed by phosphorylation of ethanolamine (Fig. 1, step 26), activation with CTP to form CDP-ethanolamine (Fig. 1, step 27), and transfer of the phosphoethanolamine moiety to DAG to form PtdEtn (Fig. 1, step 24). The Chlamydomonas CTP:phosphoethanolamine cytidylyltransferase enzyme was recently characterized by expression in E. coli and biochemical characterization of the recombinant protein (58).

Certain pathways are assumed to be present in multiple compartments. For example, PtdGro biosynthesis is proposed to be present in the plastid, mitochondria, and ER, and genes encoding three isoforms of phosphatidylglycerophosphate synthase are present in the genome. Two of these (Fig. 1, step 8) are expressed, as judged by EST analysis, and both proteins contain a predicted plastid or mitochondrial targeting sequence. The other isoform is predicted to be cytosolic but is not represented in the EST database. This finding may correlate with the fact that PtdGro is predicted to be present at a much lower abundance in the cytosol than in the plastid (19); thus, the gene encoding the cytosolic activity might not be as highly expressed as those directing PtdGro synthesis in the organelles. It is also noteworthy that we were unable to identify a gene for cardiolipin synthase, an enzyme typically present in the mitochondria of eukaryotes.

(iv) Fatty acid desaturases. In addition to the genes involved in the synthesis of the glycerolipids themselves, fatty acid desaturase genes were identified and annotated. The FAD gene complement of Arabidopsis has been well characterized at the genetic and biochemical levels (33), and orthologs are present in the Chlamydomonas genome, as outlined in Fig. 2. Of note are the putative extraplastidic 6- and 3-desaturase genes (Fig. 2, steps 38 and 39), as well as predicted plastidic orthologs of the Arabidopsis FAD5, FAD6, and FAD7 or FAD8 genes (33) (Fig. 2, steps 34 to 36). The plastidic 6-desaturase gene of Chlamydomonas (Fig. 2, step 35) was identified previously in a mutant screening for high-chlorophyll fluorescence mutants and subsequently was cloned and characterized (41, 44). We were unable to assign candidate genes for the 4-desaturase involved in the generation of the unusual fatty acid 16:4^4,7,10,13 (carbons:double bonds^positions) in the plastid (Fig. 2, step 37) or the 5-desaturase involved in 18:3^5,9,12 and 18:4^5,9,12,15 synthesis in the ER (Fig. 2) (40).

BTA1_Cr is a bifunctional protein sufficient for DGTS biosynthesis. The pathway of betaine lipid biosynthesis in the bacterium R. sphaeroides and the proposed involvement of the enzymes BtaA_Rs and BtaB_Rs are shown in Fig. 3. In this bacterium, the betaine lipid DGTS is formed under phosphate-limited growth conditions (5). Chlamydomonas is unusual in that its membranes are devoid of PtdCho and constitutively contain DGTS in its place. Searching of the whole-genome shotgun sequence assembly with the bacterial BtaA_Rs protein sequence (27) revealed a predicted protein (encoded by transcript C_570065) which we have named BTA1_Cr. It shows 27% identity and 40% similarity to BtaA_Rs over about 400 amino acids. In addition, portions of BTA1_Cr were similar to the bacterial BtaB_Rs protein. Indeed, examination of the genomic structure of BTA1_Cr (C_570065) revealed that the protein consists of an N-terminal domain with similarity to BtaB_Rs and a C-terminal portion with similarity to BtaA_Rs (Fig. 4A). The genomic locus is approximately 6 kb long, and the transcript is interrupted 11 times by introns (Fig. 4A). The predicted mRNA also contains a relatively long 3' untranslated region of 1.6 kb which is common to many Chlamydomonas transcripts.

View larger version (7K): [in this window] [in a new window]

FIG. 3. Reaction scheme for DGTS biosynthesis as determined in R. sphaeroides and Chlamydomonas. The bacterial reactions are carried out by individual proteins BtaA and BtaB, while Chlamydomonas contains the BTA1 protein, which is a fusion of BtaA-like and BtaB-like domains. Site-directed mutagenesis of the putative AdoMet binding motifs in the individual domains causes lesions in the pathway, as indicated by µA and µB.

View larger version (43K): [in this window] [in a new window]

FIG. 4. Identification of a BtaA-BtaB fusion protein encoded by the Chlamydomonas genome. (A) An approximately 6-kb locus (gene model C_570065) encodes a protein with N- and C-terminal domains showing similarity to the bacterial betaine lipid biosynthesis proteins BtaB and BtaA, respectively. 5' and 3' untranslated regions are indicated in grey, and exons and introns are indicated as thick and thin line segments, respectively. (B) Multiple-sequence alignment of the BTA1Cr protein and its bacterial counterparts in R. sphaeroides. The residues marked with arrowheads were targeted for alanine mutagenesis in the µA and µB proteins. The sequences of BtaBRs and BtaARs (GenBank accession numbers AAK53561 and AAK53560, respectively) were treated as a single entity here; the starting methionine of BtaARs is indicated by double underlining. The BtaBRs protein sequence is indicated by a solid line; the BtaARs protein sequence is indicated by a broken line. Grey shading and black shading indicate conserved and identical residues, respectively; dashes indicate gaps.

Figure 4B shows a sequence alignment of BTA1_Cr and the BtaA_Rs and BtaB_Rs proteins. The residues identified for BtaA_Rs as being potentially important in AdoMet binding are conserved in BTA1_Cr, and two residues in predicted AdoMet binding motif II (24) of the BtaA-like domain (V340 and D341) are indicated in Fig. 4B. To demonstrate functional homology between the two bacterial proteins and the two domains of BTA1_Cr, amino acids V340 and D341 were chosen for the mutagenesis experiments described below, because we expected that a V340A-D341A mutant would likely be incapable of binding AdoMet. This mutation would render the protein inactive in the DGHS synthesis reaction, but since the native conformation of the protein would probably be retained, the BtaB-like portion would be expected to still be active in the methylation steps. Additionally, the analogous positions in AdoMet binding motif II (24) of the BtaB-like portion (V121 and D122) were targeted for alanine mutagenesis (Fig. 4B). In this situation, it was presumed that DGHS synthesis activity would be intact but that the methyltransferase portion would be unable to bind AdoMet and therefore would be inactive. Cloning of the BTA1_Cr cDNA (GenBank AY656806 [GenBank] ) and expression in E. coli led to the accumulation of relatively large amounts of DGTS, allowing structure elucidation. Figure 5A shows a TLC separation of lipids from E. coli expressing BTA1_Cr, and it is apparent that there is a new species accumulating in the expression strain. This lipid was subsequently identified as DGTS by positive-ion fast-atom bombardment-mass spectrometry (Fig. 5B) and NMR spectroscopy. It consists of molecular species that are typical of E. coli membrane lipids; e.g., the species at m/z 710.7 is consistent with DGTS carrying 32:1 (carbons:double bonds) fatty acids, and that at m/z 736.7 is a 34:2 species of DGTS. The ¹H NMR spectrum (data not shown) exhibited resonances consistent with those previously reported (5), most notably, a strong nine-proton resonance at 3.22 ppm, which corresponds to the methyl protons of the DGTS quaternary ammonium group.

View larger version (38K): [in this window] [in a new window]

FIG. 5. BTA1Cr is sufficient to catalyze the biosynthesis of DGTS. (A) E. coli harboring pBTA1 was grown and expressed as described in Materials and Methods, and lipids were separated by TLC and visualized with iodine vapor. A culture containing empty vector pQE-31 served as a negative control. CL, cardiolipin. (B) Fast-atom bombardment-mass spectrometry in the positive-ion mode was used to confirm the identity of the putative DGTS spot on the TLC plates. Peaks at m/z 710.7 and m/z 736.7 represent the most abundant molecular species, with acyl chain lengths and total double bonds (carbons:double bonds) of 32:1 and 34:2, respectively.

Kinetic analysis of BTA1_Cr. To characterize the activity of the BTA1_Cr protein, we expressed the gene in E. coli, broke the cells by sonication, and assayed the incorporation of [1-¹⁴C]AdoMet into labeled DGHS, partially methylated DGHS derivatives, and the end product DGTS. In Fig. 6, the pH-activity profile and the determination of the AdoMet K_m for the overall reaction of BTA1_Cr are shown. The relative stoichiometries of the methylation products change with increasing pHs (Fig. 6A), indicating a difference in the pH optima between the BtaA-like and the BtaB-like domains. Figure 6B shows the quantification of the total counts in each lane of Fig. 6A, plotted against the pH; this analysis indicates that the pH optimum of the overall reaction is 7.2. Figure 6C shows a plot of the rate of overall product formation against substrate concentration; this analysis was used to determine, by direct fitting of a hyperbola to the experimental data, that the apparent K_m for AdoMet was 16 µM.

View larger version (26K): [in this window] [in a new window]

FIG. 6. Biochemical properties and site-directed mutagenesis of BTA1Cr. (A) TLC of products of BTA1Cr at various pHs. Cell lysates were prepared and adjusted to the proper pHs as described in Materials and Methods, followed by incubation with [1-14C]AdoMet (4.2 µM) for 30 min. Products were extracted and separated by TLC, followed by exposure to a PhosphorImager screen. DGM/DS, a mixture of mono- and di-N-methylated DGHS. (B) The radioactivity in each lane of the image in panel A was quantified with the ImageQuant software package and used to determine the pH optimum for DGHS synthase activity. (C) The apparent Km for AdoMet was determined at pH 7.2; the concentrations of AdoMet were varied. The apparent Km was determined by direct fitting of a hyperbola to the experimental data in the Origin software package.

Site-directed mutagenesis of AdoMet binding domains of BTA1_Cr. To corroborate the relatedness of the two domains of BTA1_Cr and the respective bacterial proteins, site-directed mutagenesis was used to change residues predicted to be critical for AdoMet binding in the active sites of the BtaA-like and BtaB-like domains. These were designated BTA1_CrµA and BTA1_CrµB, respectively (Fig. 3). The BTA1_CrµA protein was inactive in the synthesis of DGHS, as shown in Fig. 7, second lane, with only background levels of label in the DGTS or DGHS regions of the chromatogram. BTA1_CrµB, however, produced DGHS (Fig. 7, third lane), as confirmed by cochromatography with the product of a strain carrying btaA_Rs (Fig. 3) on a low-copy-number plasmid, which caused the accumulation of DGHS (Fig. 7, fourth lane). Figure 7, fifth lane, shows DGTS accumulating in the pBTA1-containing E. coli strain, and Fig. 7, sixth lane, shows that the BtaB-like methyltransferase domain in pBTA1-µA was still capable of methylating DGHS produced by BtaA_Rs. These results establish the functions of the two domains of BTA1_Cr as being comparable to those of the individual bacterial orthologs and corroborate the hypothesis that BTA1_Cr is capable of carrying out all reactions necessary for DGTS biosynthesis in Chlamydomonas.

View larger version (109K): [in this window] [in a new window]

FIG. 7. Analysis of BtaA-like and BtaB-like domains of BTA1Cr by site-directed mutagenesis. E. coli harboring the combinations of plasmids indicated above the lanes was labeled with [1-14C]methionine. The lipids were separated by TLC, followed by exposure to a PhosphorImager screen.

Top Abstract Introduction Materials and Methods Results Discussion References

 
With the exception of our case study of BTA1_Cr, all of our conclusions are based on an in silico analysis of genome sequences of Chlamydomonas. We present an annotation of glycerolipid biosynthetic genes and a subsequent metabolic pathway reconstruction, which was guided in part by our general knowledge of lipid biosynthesis in Arabidopsis and other plants. Genome comparisons between less well-studied organisms, such as Chlamydomonas, and more well-studied model organisms, such as Arabidopsis, at least with regard to lipid metabolism, provide great opportunities to rapidly assess the evolution or adaptation of, in this case, Chlamydomonas; they also permit one to focus on the essential differences, e.g., the absence of PtdCho and the biosynthesis of DGTS in Chlamydomonas. However, one should not forget that annotation by an in silico analysis primarily serves to build hypotheses, which subsequently need to be rigorously corroborated to gain generally accepted knowledge. For example, the absence of a certain enzyme or pathway does not mean that alternative pathways do not exist. Likewise, predicting the subcellular localizations of proteins from signal sequences is possible only with limitations. Keeping these caveats in mind, we cautiously draw the conclusions discussed below, which hopefully will stimulate and guide further membrane lipid research with Chlamydomonas.

Chlamydomonas lipid metabolism is less complex than that of flowering plants. During the course of bioinformatic analysis and annotation, several themes became apparent. First, the core pathways of fatty acid biosynthesis and glycerolipid assembly in Chlamydomonas appear to be simpler than those in Arabidopsis, reflected both in the presence and/or in the absence of certain pathways and in the apparent sizes of the gene families that represent the various activities. A case in point is that of galactolipid synthesis. In the Chlamydomonas genome, we have identified only one MGDG synthase gene (MGD1) and one DGDG synthase gene (DGD1), as opposed to three MGD1 isoform-encoding genes (3) and two DGD1 isoform-encoding genes (26) in the Arabidopsis genome. This phenomenon may be thought of as being parallel to the lack of PtdCho and its presumed replacement by the nonphosphorous analog DGTS in Chlamydomonas. In Arabidopsis, phosphate deprivation has been shown to up-regulate the expression of MGD2, MGD3, and DGD2, concomitant with a loss of PtdCho and an accumulation of DGDG in extraplastidic membranes (10). This scenario has also been observed in phosphate-starved oat, where DGDG, presumably produced via the phosphate starvation-inducible biosynthetic pathway, replaces a large proportion of PtdCho in the plasma membrane (2). This phenomenon is presumed to be an adaptation to phosphate limitation, in that a nonphosphorous lipid is substituting for a phospholipid, thus allowing reallocation of the limiting nutrient into more critical components, such as nucleic acids. Given that Chlamydomonas apparently has no need to replace PtdCho with DGDG due to the presence of DGTS, the phosphate-inducible pathway of DGDG biosynthesis may be expendable and has been lost during the course of evolution, or it may simply have evolved only in flowering plants.

Potential for dual targeting of proteins involved in lipid metabolism. A second theme is apparent in the biosynthesis of fatty acids, in that some of the core components of this process may be dually targeted to both the plastid and the mitochondrion. Several lines of evidence support this hypothesis. First, it is known from studies of isolated organelles of flowering plants that fatty acid synthesis occurs in both plastids and mitochondria (54), the former being responsible for the majority of fatty acid biosynthesis to supply precursors for membrane lipid synthesis and the latter being a function of the biosynthesis of lipoic acid, a critical prosthetic group for certain mitochondrial enzymes (49). Several genes encoding enzymes that are central to fatty acid synthesis, e.g., components of the multimeric acetyl-CoA carboxylase and the type II fatty acid synthase, as well as ACPs, are potentially represented by only one gene (two genes in the case of ACPs) in the Chlamydomonas genome. Since the activities are present in multiple compartments but may be the products of a single gene, these proteins may be imported by both plastids and mitochondria; this phenomenon has a growing number of precedents, especially for proteins involved in core metabolic processes, such as nucleotide biosynthesis and DNA replication (7, 55).

In agreement with these observations, the Chlamydomonas proteins in question have a relatively high probability of dual targeting to mitochondria and plastids, as judged by TargetP analysis. The recent finding of a potentially dually targeted multifunctional acetyl-CoA carboxylase in grasses (15) also lends credence to this hypothesis. Additionally, Beisson et al. (4) were unable to predict candidates for the mitochondrial 3-ketoacyl-ACP-reductase and 3-hydroxyacyl-CoA-dehydratase activities of Arabidopsis. Given that these activities have been demonstrated implicitly by the fact that mitochondria can synthesize fatty acids, it is reasonable to posit that dual targeting of these proteins could be operative in both Arabidopsis and Chlamydomonas, although direct experimental evidence is currently lacking.

BTA1_Cr is a bifunctional enzyme involved in DGTS biosynthesis. BTA1_Cr was identified based on similarities to the bacterial betaine lipid synthesis proteins BtaA_Rs and BtaB_Rs, encoding the presumed AdoMet:DAG 3-amino-3-carboxypropyltransferase and the DGHS methylase, respectively (Fig. 3). To experimentally verify the predicted enzymatic activities of BTA1_Cr, we expressed the BTA1_Cr cDNA (GenBank AY656806 [GenBank] ) in E. coli. This procedure resulted in DGTS biosynthesis from the expression of a single protein, thus confirming that BTA1_Cr catalyzes all of the reactions specific to the pathway. The production of the BtaA-like portion of BTA1_Cr by itself in E. coli was expected to result in DGHS accumulation, as was shown for BtaA_Rs (Fig. 7, fourth lane); however, we were unable to show that the truncated protein has DGHS synthesis activity in E. coli (data not shown).

As an alternative way of revealing the functions of the two domains of BTA1_Cr, we altered residues in the predicted AdoMet binding sites of the BtaA-like and BtaB-like domains. The results of this experiment were informative on several levels. First, the fact that the BTA1_Cr V340A-D341A mutant (BTA1_CrµA) was inactive in the synthesis of DGHS and DGTS lends credence to the model that the BtaA-like domain binds AdoMet in a methyltransferase fold. Given the structure of the cognate binding site of methyltransferases, V340 is expected to make favorable hydrophobic interactions with the adenine moiety of AdoMet, and D341 is proposed to form hydrogen bonds to the 2' and 3' hydroxyls of the ribose portion. Mutation of these residues to alanine rendered the DGHS synthase function of BTA1_Cr inactive or at least compromised it to below the limits of detection. The DGHS trimethylase function was left unaffected, given that the BTA1_CrµA protein was still capable of methylating DGHS produced by simultaneous expression of the bacterial btaA_Rs gene (Fig. 7, sixth lane). This finding provided an additional piece of information regarding the sequence of reactions, showing that the BtaB-like domain of BTA1_Cr is able to take DGHS from the membrane as a substrate, rather than that it is transferred directly from the active site of the BtaA-like domain, as would be the case for substrate channeling. These data are in agreement with the findings of in vitro assays conducted with isolated C. reinhardtii membranes (29) as well as the results presented in Fig. 6A, showing an accumulation of a transient pool of DGHS in in vitro assays.

Enzyme mechanisms for DGTS biosynthesis are conserved across kingdoms. BTA1_Cr activity was measured in a cell-free system, relying on endogenously formed DAG, the presumed lipid substrate, and exogenous radiolabeled AdoMet. The analysis was somewhat complicated by the fact that DGHS synthase and methyltransferase activities were present on the same polypeptide; thus, multiple products were formed. However, measurement of all products on the same TLC plate (Fig. 6A) made this a trivial point, in that the sum of DGHS and its methylated products gave the total flux through the BtaA-like domain, and this quantity was used in the analyses shown in Fig. 6B and C. In fact, Fig. 6A is informative as to the activity of the BtaB-like (N-methyltransferase) portion, showing that the formation of the mono- and dimethylated DGHS intermediates is preferred as the pH increases and that the final methylation (forming DGTS) is favored at an even higher pH (8). The pH dependence of these subsequent methylation reactions may reflect a change in the pK_a values of amine species with higher degrees of methylation. The apparent K_m for recombinant BTA1_Cr activity is presumably directly comparable to that for the activity that was measured in C. reinhardtii membranes (29). However, the value for the recombinant enzyme (16 µM) was about fivefold lower than that for the native enzyme, indicating that the membrane composition and environment of the enzyme may affect the affinity for AdoMet of the DGHS synthase module. This scenario is not surprising, considering that a thorough kinetic description would require both AdoMet and DAG concentrations to be varied; i.e., at higher DAG concentrations (as might occur in E. coli membranes), the apparent K_m of the BTA1_Cr A domain for AdoMet might be lower. The question of the true K_m remains unanswered given our current inability to reconstitute BTA1_Cr into the defined lipid environment of a synthetic liposome.

Taken together, the results presented here show that the enzymes and mechanisms of DGTS biosynthesis are conserved between eukaryotes and prokaryotes, with the exception that the eukaryotic activities are fused into a single polypeptide. This latter phenomenon is common among metabolic enzymes, such as urease, which consists of three separate structural components in Klebsiella aerogenes that are fused together on a single polypeptide in legumes (20), and acetyl-CoA carboxylase, which is present as a multimeric complex in bacteria but consists of a single multifunctional polypeptide in eukaryotes (30). Apparently, the same regulatory advantages that are in play in those systems are important in DGTS biosynthesis as well; i.e., having one gene to encode the machinery for the entire pathway presumably alleviates the coordination required for the proper expression of multiple individual genes.

Conclusions. In this study, we identified a set of genes encoding products that are likely to be involved in membrane glycerolipid biosynthesis in Chlamydomonas. However, the assembly of a biological membrane is more than the sum of its biosynthetic reactions, and there are undoubtedly layers of complexity which this study fails to reach. One area in which this gene set is lacking is lipid transport, which is likely to be critical in maintaining the proper lipid composition of a given membrane. For example, the situation of a lipid being synthesized in one membrane and transported to another must be accounted for when one is devising a mechanistic description of membrane biogenesis. Likewise, simply knowing the genes involved in PtdEtn and DGTS biosynthesis (or that of any other set of lipids in a given membrane) implies nothing about how the final stoichiometric ratio between them is regulated so as to form a functional membrane with the correct biophysical properties. Given the apparent lack of redundancy in the pathways described above, the absence of complicating factors, such as tissue specificity, and the potential for functional analysis by gene silencing, Chlamydomonas is poised to be a useful model for studying how plant cells sense and control the flux of precursors into different polar lipid biosynthesis pathways in order to produce a functional membrane.

 
We thank Beverly Chamberlin from the MSU Mass Spectrometry Facility for help with mass spectrometric analyses.

This work was supported in part by grants from NSF (MCB 0109912 to C.B. and MCB 9982600 to B.B.S.) and from the MSU Center for Novel Plant Products and Technologies (to C.B.).

 
* Corresponding author. Mailing address: 215 Biochemistry Bldg., Michigan State University, East Lansing, MI 48824. Phone: (517) 355-1609. Fax: (517) 353-9334. E-mail: benning{at}msu.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
2
2. Andersson, M. X., M. H. Stridh, K. E. Larsson, C. Liljenberg, and A. S. Sandelius. 2003. Phosphate-deficient oat replaces a major portion of the plasma membrane phospholipids with the galactolipid digalactosyldiacylglycerol. FEBS Lett. 537:128-132.[CrossRef][Medline]
3
3. Awai, K., E. Marechal, M. A. Block, D. Brun, T. Masuda, H. Shimada, K. Takamiya, H. Ohta, and J. Joyard. 2001. Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 98:10960-10965.[Abstract/Free Full Text]
4
4. Beisson, F., A. J. Koo, S. Ruuska, J. Schwender, M. Pollard, J. J. Thelen, T. Paddock, J. J. Salas, L. Savage, A. Milcamps, V. B. Mhaske, Y. Cho, and J. B. Ohlrogge. 2003. Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol. 132:681-697.
5
5. Benning, C., Z. H. Huang, and D. A. Gage. 1995. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch. Biochem. Biophys. 317:103-111.[CrossRef][Medline]
6
6. Bolognese, C. P., and P. McGraw. 2000. The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phosphoethanolamine N-methyltransferase. Plant Physiol. 124:1800-1813.[Abstract/Free Full Text]
7
7. Chabregas, S. M., D. D. Luche, L. P. Farias, A. F. Ribeiro, M. A. van Sluys, C. F. M. Menck, and M. C. Silva. 2001. Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts. Plant Mol. Biol. 46:639-650.[CrossRef][Medline]
8
8. Davies, J. P., F. H. Yildiz, and A. Grossman. 1996. Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J. 15:2150-2159.[Medline]
9
9. Dörmann, P., I. Balbo, and C. Benning. 1999. Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD1. Science 284:2181-2184.[Abstract/Free Full Text]
10
10. Dörmann, P., and C. Benning. 2002. Galactolipids rule in seed plants. Trends Plant Sci. 7:112-118.[CrossRef][Medline]
11
11. El Maanni, A., G. Dubertret, M. J. Delrieu, O. Roche, and A. Tremolieres. 1998. Mutants of Chlamydomonas reinhardtii affected in phosphatidylglycerol metabolism and thylakoid biogenesis. Plant Physiol. Biochem. 36:609-619.[CrossRef]
12
12. Emanuelsson, O., H. Nielsen, S. Brunak, and G. von Heijne. 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300:1005-1016.[CrossRef][Medline]
13
13. Emanuelsson, O., H. Nielsen, and G. von Heijne. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8:978-984.[Medline]
14
14. Essigmann, B., S. Güler, R. A. Narang, D. Linke, and C. Benning. 1998. Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95:1950-1955.[Abstract/Free Full Text]
15
15. Focke, M., E. Gieringer, S. Schwan, L. Jansch, S. Binder, and H. P. Braun. 2003. Fatty acid biosynthesis in mitochondria of grasses: malonyl-coenzyme A is generated by a mitochondrial-localized acetyl-coenzyme A carboxylase. Plant Physiol. 133:875-884.[Abstract/Free Full Text]
16
16. Frentzen, M. 2004. Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation. Curr. Opin. Plant Biol. 7:270-276.[CrossRef][Medline]
17
17. Fuhrmann, M., A. Stahlberg, E. Govorunova, S. Rank, and P. Hegemann. 2001. The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses. J. Cell Sci. 114:3857-3863.[Abstract/Free Full Text]
18
18. Giroud, C., and W. Eichenberger. 1989. Lipids of Chlamydomonas reinhardtii—incorporation of [C-14] acetate, [C-14] palmitate and [C-14] oleate into different lipids and evidence for lipid-linked desaturation of fatty acids. Plant Cell Physiol. 30:121-128.[Abstract/Free Full Text]
19
19. Giroud, C., A. Gerber, and W. Eichenberger. 1988. Lipids of Chlamydomonas reinhardtii. Analysis of molecular species and intracellular site(s) of biosynthesis. Plant Cell Physiol. 29:587-595.
20
20. Goldraij, A., L. J. Beamer, and J. C. Polacco. 2003. Interallelic complementation at the ubiquitous urease coding locus of soybean. Plant Physiol. 132:1801-1810.[Abstract/Free Full Text]
21
21. Grossman, A. R., E. E. Harris, C. Hauser, P. A. Lefebvre, D. Martinez, D. Rokhsar, J. Shrager, C. D. Silflow, D. Stern, O. Vallon, and Z. Zhang. 2003. Chlamydomonas reinhardtii at the crossroads of genomics. Eukaryot. Cell 2:1137-1150.[Free Full Text]
22
22. Harris, E. H. 1989. The Chlamydomonas source book. Academic Press, Inc., New York, N.Y.
23
23. Hofmann, M., and W. Eichenberger. 1996. Biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N methylation. J. Bacteriol. 178:6140-6144.[Abstract/Free Full Text]
24
24. Kagan, R. M., and S. Clarke. 1994. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310:417-427.[CrossRef][Medline]
25
25. Kathir, P., M. LaVoie, W. J. Brazelton, N. A. Haas, P. A. Lefebvre, and C. D. Silflow. 2003. Molecular map of the Chlamydomonas reinhardtii nuclear genome. Eukaryot. Cell 2:362-379.[Abstract/Free Full Text]
26
26. Kelly, A. A., and P. Dörmann. 2002. DGD2, an Arabidopsis gene encoding a UDP-galactose-dependent digalactosyldiacylglycerol synthase is expressed during growth under phosphate-limiting conditions. J. Biol. Chem. 277:1166-1173.[Abstract/Free Full Text]
27
27. Klug, R. M., and C. Benning. 2001. Two enzymes of diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. USA 98:5910-5915.[Abstract/Free Full Text]
28
28. Lonsdale, D. 1998. Update on the Commission on Plant Gene Nomenclature (CPGN) and the Mendel database. Plant Mol. Biol. Rep. 16:215-216.[CrossRef]
29
29. Moore, T. S., Z. Du, and Z. Chen. 2001. Membrane lipid biosynthesis in Chlamydomonas reinhardtii. In vitro biosynthesis of diacylglyceryltrimethylhomoserine. Plant Physiol. 125:423-429.[Abstract/Free Full Text]
30
30. Nikolau, B. J., J. B. Ohlrogge, and E. S. Wurtele. 2003. Plant biotin-containing carboxylases. Arch. Biochem. Biophys. 414:211-222.[CrossRef][Medline]
31
31. Niyogi, K. K. 1999. Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:333-359.[CrossRef]
32
32. Nuccio, M. L., M. J. Ziemak, S. A. Henry, E. A. Weretilnyk, and A. D. Hanson. 2000. cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme. J. Biol. Chem. 275:14095-14101.[Abstract/Free Full Text]
33
33. Ohlrogge, J., and J. Browse. 1995. Lipid biosynthesis. Plant Cell 7:957-970.[CrossRef][Medline]
34
34. Pineau, B., J. Girard-Bascou, S. Eberhard, Y. Choquet, A. Tremolieres, C. Gerard-Hirne, A. Bennardo-Connan, P. Decottignies, S. Gillet, and F. A. Wollman. 2004. A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii. Eur. J. Biochem. 271:329-338.[Medline]
35
35. Riekhof, W. R., M. E. Ruckle, T. A. Lydic, B. B. Sears, and C. Benning. 2003. The sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1. Plant Physiol. 133:864-874.[Abstract/Free Full Text]
36
36. Rontein, D., I. Nishida, G. Tashiro, K. Yoshioka, W. I. Wu, D. R. Voelker, G. Basset, and A. D. Hanson. 2001. Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme. J. Biol. Chem. 276:35523-35529.[Abstract/Free Full Text]
37
37. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
38
38. Sanda, S., T. Leustek, M. Theisen, M. Garavito, and C. Benning. 2001. Recombinant Arabidopsis SQD1 converts UDP-glucose and sulfite to the sulfolipid head precursor UDP-sulfoquinovose in vitro. J. Biol. Chem. 276:3941-3946.[Abstract/Free Full Text]
39
39. Sato, N. 1988. Dual role of methionine in the biosynthesis of diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii. Plant Physiol. 86:931-934.[Abstract/Free Full Text]
40
40. Sato, N. 1992. Betaine lipids. Bot. Mag. Tokyo 105:185-197.[CrossRef]
41
41. Sato, N., S. Fujiwara, A. Kawaguchi, and M. Tsuzuki. 1997. Cloning of a gene for chloroplast omega 6 desaturase of a green alga, Chlamydomonas reinhardtii. J. Biochem. 122:1224-1232.[Abstract/Free Full Text]
42
42. Sato, N., and N. Murata. 1991. Transition of lipid phase in aqueous dispersions of diacylglyceryltrimethylhomoserine. Biochim. Biophys. Acta 1082:108-111.[Medline]
43
43. Sato, N., K. Sugimoto, A. Meguro, and M. Tsuzuki. 2003. Identification of a gene for UDP-sulfoquinovose synthase of a green alga, Chlamydomonas reinhardtii, and its phylogeny. DNA Res. 10:229-237.[Abstract]
44
44. Sato, N., M. Tsuzuki, Y. Matsuda, T. Ehara, T. Osafune, and A. Kawaguchi. 1995. Isolation and characterization of mutants affected in lipid metabolism of Chlamydomonas reinhardtii. Eur. J. Biochem. 230:987-993.[Medline]
45
45. Seras, M., J. Garnier, A. Tremolieres, and D. Guyon. 1989. Lipid biosynthesis in cells of the wild-type and of 2 photosynthesis mutants of Chlamydomonas reinhardtii. Plant Physiol. Biochem. 27:393-399.
46
46. Shrager, J., C. Hauser, C. W. Chang, E. H. Harris, J. Davies, J. McDermott, R. Tamse, Z. Zhang, and A. R. Grossman. 2003. Chlamydomonas reinhardtii genome project. A guide to the generation and use of the cDNA information. Plant Physiol. 131:401-408.
47
47. Silflow, C. D., and P. A. Lefebvre. 2001. Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiol. 127:1500-1507.
48
48. Sineshchekov, O. A., K. H. Jung, and J. L. Spudich. 2002. Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 99:8689-8694.[Abstract/Free Full Text]
49
49. Somerville, C., J. Browse, J. G. Jaworski, and J. B. Ohlrogge. 2000. Lipids, p. 457-527. In B. B. Buchanan, W. Gruissem, and R. L. Jones (ed.), Biochemsitry and molecular biology of plants. American Society of Plant Physiologists, Rockville, Md.
50
50. Tam, L. W., and P. A. Lefebvre. 1993. Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics 135:375-384.[Abstract]
51
51. The Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815.[CrossRef][Medline]
52
52. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
53
53. Voelker, D. R. 1997. Phosphatidylserine decarboxylase. Biochim. Biophys. Acta 1348:236-244.[Medline]
54
54. Wada, H., D. Shintani, and J. Ohlrogge. 1997. Why do mitochondria synthesize fatty acids? Evidence for involvement in lipoic acid production. Proc. Natl. Acad. Sci. USA 94:1591-1596.
55
55. Wall, M. K., L. A. Mitchenall, and A. Maxwell. 2004. Arabidopsis thaliana DNA gyrase is targeted to chloroplasts and mitochondria. Proc. Natl. Acad. Sci. USA 101:7821-7826.[Abstract/Free Full Text]
56
56. Wu-Scharf, D., B. R. Jeong, C. M. Zhang, and H. Cerutti. 2000. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science 290:1159-1162.[Abstract/Free Full Text]
57
57. Xu, C., J. Fan, W. Riekhof, J. E. Froehlich, and C. Benning. 2003. A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis. EMBO J. 22:2370-2379.[CrossRef][Medline]
58
58. Yang, W., C. B. Mason, S. V. Pollock, T. Lavezzi, J. V. Moroney, and T. S. Moore. 2004. Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase. Biochem. J. 382:51-57.[CrossRef][Medline]
59
59. Yu, B., C. Xu, and C. Benning. 2002. Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proc. Natl. Acad. Sci. USA 99:5732-5737.[Abstract/Free Full Text]

---

Eukaryotic Cell, February 2005, p. 242-252, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.242-252.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Awai, K., Watanabe, H., Benning, C., Nishida, I. (2007). Digalactosyldiacylglycerol is Required for Better Photosynthetic Growth of Synechocystis sp. PCC6803 Under Phosphate Limitation. Plant Cell Physiol 48: 1517-1523 [Abstract] [Full Text]  
• Sato, N., Moriyama, T. (2007). Genomic and Biochemical Analysis of Lipid Biosynthesis in the Unicellular Rhodophyte Cyanidioschyzon merolae: Lack of a Plastidic Desaturation Pathway Results in the Coupled Pathway of Galactolipid Synthesis. Eukaryot Cell 6: 1006-1017 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Riekhof, W. R. Articles by Benning, C. Search for Related Content PubMed PubMed Citation Articles by Riekhof, W. R. Articles by Benning, C.