Endoplasmic reticulum acyltransferase withprokaryotic substrate preference contributes totriacylglycerol assembly in ChlamydomonasYeongho Kima,b, Ee Leng Ternga,b, Wayne R. Riekhofa, Edgar B. Cahoonb,c, and Heriberto Ceruttia,b,1

aSchool of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588; bCenter for Plant Science Innovation, University of Nebraska-Lincoln,Lincoln, NE 68588; and cDepartment of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588

Edited by Krishna K. Niyogi, Howard Hughes Medical Institute and University of California, Berkeley, CA, and approved January 3, 2018 (received for reviewSeptember 9, 2017)

Understanding the unique features of triacylglycerol (TAG) metab-olism in microalgae may be necessary to realize the full poten-tial of these organisms for biofuel and biomaterial production. Inthe unicellular green alga Chlamydomonas reinhardtii a chloro-plastic (prokaryotic) pathway has been proposed to play a majorrole in TAG precursor biosynthesis. However, as reported here, C.reinhardtii contains a chlorophyte-specific lysophosphatidic acidacyltransferase, CrLPAAT2, that localizes to endoplasmic reticulum(ER) membranes. Unlike canonical, ER-located LPAATs, CrLPAAT2prefers palmitoyl-CoA over oleoyl-CoA as the acyl donor sub-strate. RNA-mediated suppression of CrLPAAT2 indicated that theenzyme is required for TAG accumulation under nitrogen depri-vation. Our findings suggest that Chlamydomonas has a distinctglycerolipid assembly pathway that relies on CrLPAAT2 to gener-ate prokaryotic-like TAG precursors in the ER.

LPAAT | lipid droplets | triacylglycerol metabolism | algae | biofuels

Triacylglycerol (TAG) is a major storage lipid in most eukary-otes and a precursor for biodiesel production (1, 2). Some

microalgae have recently gained attention because they can accu-mulate large amounts of TAGs and potentially serve as feedstockfor biofuel production (2–4). However, despite current advances,our understanding of algal lipid metabolism is still fairly lim-ited and generally based on insights from land plants, even forthe well-studied model system Chlamydomonas reinhardtii (3,4). However, algal metabolism appears to have some distinctfeatures (4, 5) whose understanding may be required for thebiotechnological improvement of algal strains.

During the biogenesis of complex lipids in plants, fatty acidssynthesized de novo in the chloroplast can be assembled intoglycerolipids by the prokaryotic (plastidial) pathway or theycan be exported to the endoplasmic reticulum (ER), enteringthe eukaryotic pathway of glycerolipid assembly. Glycerolipidssynthesized by the prokaryotic pathway carry a 16-carbon acylchain at the sn-2 position of the glycerol backbone, whereasglycerolipids assembled by the eukaryotic pathway contain an18-carbon acyl chain at the same position (6). This distinc-tion is caused by differences in the substrate specificity oflysophosphatidic acid acyltransferases (LPAATs). Chloroplast-localized LPAATs mainly use palmitoyl-ACP (C16:0-ACP)as the acyl donor to generate sn-2-C16:0-phosphatidic acid(PA) (7, 8) while those in the ER prefer using oleoyl-CoA (C18:1-CoA) to synthesize sn-2-C18:1-PA (9, 10). Theseobservations have been recapitulated in numerous land plants(7–11).

In plant seeds, the assembly of storage TAGs occurs in theER, having C18 esterified at the sn-2 position of their glycerolbackbone (11). Moreover, in plants, mammals, and fungi, micro-scopic observations of TAG-containing lipid droplets (LDs)and the subcellular location of major enzymes involved in thefinal step of TAG synthesis, such as acyl-CoA:diacylglycerolacyltransferases (DGATs) and phospholipid:diacylglycerol acyl-

transferase (PDAT), are also consistent with TAG assembly tak-ing place in the ER (11–13).

In contrast, in C. reinhardtii, TAGs accumulated under nitro-gen deprivation mostly have C16 at their sn-2 position and it hasbeen hypothesized that the plastidial pathway plays a major role inTAG synthesis (4, 14, 15). Since a canonical, ER-targeted LPAATwas not identified in the C. reinhardtii genome (16), the plas-tidial chlorophyte-specific (Cr)LPAAT1 has been suggested toparticipate actively in generating precursors for TAG accumula-tion (15). Moreover, light and electron microscopy revealed LDsin both the cytosol and the chloroplast of nutrient-starved C. rein-hardtii, although plastid-located large LDs were observed only instarchless mutants deprived of nitrogen under mixotrophic con-ditions or in wild-type strains under special environmental con-ditions such as saturating light (17, 18). These findings, togetherwith the predicted subcellular location of major enzymes of TAGbiosynthesis (3, 4), support the involvement of both the prokary-otic and eukaryotic pathways in algal TAG assembly, but theircontributions may vary depending on cultivation conditions andstrain genotype. Additionally, LPAATs have not been character-ized in detail in microalgae and it is not certain that the plantparadigm regarding LPAAT substrate specificity in the two path-ways applies universally to algal species.

Here we demonstrate that C. reinhardtii contains a uniqueLPAAT, encoded by Cre17.g738350 and termed CrLPAAT2,which seems to be restricted to the chlorophytes. CrLPAAT2 islocalized to the ER but, like prokaryotic acyltransferases, prefersC16:0-CoA over C18:1-CoA as the substrate. RNA-mediated

Significance

The acyl chain composition of Chlamydomonas triacylglyc-erols (TAGs) suggests that they are assembled from prokary-otic precursors, proposed to be synthesized in the chloroplast.However, in most eukaryotes, the endoplasmic reticulum (ER)appears to be the main organelle for storage TAG biosyn-thesis. Interestingly, Chlamydomonas reinhardtii has a dis-tinct lysophosphatidic acid acyltransferase that localizes to theER but resembles prokaryotic lysophosphatidic acid acyltrans-ferases (LPAATs) in its substrate preference. Thus, Chlamy-domonas and related green algae, unlike land plants, cansynthesize “prokaryotic” acyl-lipids in the ER, with intriguingimplications for biotechnological applications.

Author contributions: Y.K. and H.C. designed research; Y.K. and E.L.T. performedresearch; Y.K., E.L.T., W.R.R., E.B.C., and H.C. analyzed data; and Y.K. and H.C. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1 To whom correspondence should be addressed. Email: hcerutti1@unl.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1715922115/-/DCSupplemental.

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silencing of CrLPAAT2 indicated that the enzyme is requiredfor TAG accumulation under nitrogen deprivation in photoau-totrophic conditions. Thus, Chlamydomonas (and related greenalgae) appears to rely on CrLPAAT2 to generate prokaryotic-like TAG species in the ER.

ResultsSubstantial TAG accumulation is triggered by nutritional stressin many microalgae (2, 19). However, somewhat unexpect-edly, systems-level approaches revealed that nitrogen-depletedChlamydomonas showed up-regulation of few genes involvedin acyl-lipid metabolism (20–22). Moreover, several genes pre-sumably involved in de novo fatty acid synthesis appeared tobe down-regulated under these conditions, despite an increasedcarbon flux toward TAG biosynthesis (14). The transcript abun-dance of Cre17.g738350, encoding CrLPAAT2, remained rel-atively stable under nitrogen deprivation in mixotrophic con-ditions (20, 21) but increased slightly (see below) under thesame stress in photoautotrophic conditions. Despite this mod-est change in gene expression, given some distinctive features ofthe predicted CrLPAAT2 enzyme, we decided to investigate itsrole in acyl-lipid metabolism under nutritional deprivation.

CrLPAAT2 Has Unique Structural Features and Belongs to a Chloro-phyte-Specific Clade. The predicted amino acid sequence ofCre17.g738350, like conventional LPAATs, contains a LPAAT/AGPAT domain (PANTHER10434) identified by the Inter-proScan program. ChloroP and PredAlgo algorithms suggestedthat CrLPAAT2 does not have a chloroplast transit peptide(Fig. 1A and SI Appendix, Table S1). However, analyses oftransmembrane domains (TMs) by TMHMM (transmembranehidden Markov model) and of catalytic sites by InterproScanrevealed that the N-terminal half of CrLPAAT2 is fairly sim-ilar to the central region of chloroplast LPAATs (8, 15) (Fig.1A and SI Appendix, Fig. S1). In contrast, the C-terminal halfof CrLPAAT2 contains two predicted TMs, a structure simi-lar to that of eukaryotic LPAATs such as AtLPAT2 from Ara-bidopsis thaliana and LAT1 from Limnanthes douglasii (9, 23)(Fig. 1A and SI Appendix, Fig. S1). Indeed, CrLPAAT2 seemsto have a chimeric structure, combining domains from plastidialand eukaryotic LPAATs.

To gain insight into the evolutionary origin of CrLPAAT2,phylogenetic analyses were performed with available LPAAT-related algal sequences and those from assorted eukaryotesand eubacteria (SI Appendix, Fig. S2). Interestingly, CrLPAAT2belongs to a well-supported clade containing exclusively chloro-phyte proteins; and this clade was clearly distinguished fromthose containing eukaryotic LPAATs (LPAT2-5s), eukaryoticacyl-CoA:lysophospholipid acyltransferases (LPCATs/LPEATs),or plastidial LPAATs. Thus, CrLPAAT2 appears to represent adistinctive group of algal LPAATs, structurally as well as phy-logenetically divergent from well-characterized eukaryotic andprokaryotic LPAATs.

CrLPAAT2 Localizes to the Endoplasmic Reticulum. To begin assess-ing CrLPAAT2 function we examined its subcellular loca-tion. The CrLPAAT2 coding sequence was fused in frame atthe 5′ end of the mCherry fluorescent protein sequence (24).This transgene, under the control of the PsaD promoter, wasthen introduced by electroporation into C. reinhardtii CC124.CrLPAAT2-mCherry was found to localize mainly in the cytosolof Chlamydomonas, by both live-cell and immunofluorescenceimaging (Fig. 1B and SI Appendix, Fig. S3). NontransgenicCC124 was used as a negative control, to verify the absence ofany background signal in the mCherry channel.

Bioinformatic analyses suggested that CrLPAAT2 is targetedto the secretory pathway (SI Appendix, Table S1). Indeed, byusing an ER-tracker dye (BODIPY FL Glibenclamide) (25), we

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Fig. 1. Schematic diagrams of LPAATs and subcellular localization ofCrLPAAT2. (A) Schematic protein diagrams indicating key domains: chloro-plast transit peptide (red), LPAAT catalytic domain (black), and transmem-brane domains (gray). (B) Subcellular localization of the CrLPAAT2-mCherryfusion protein. Transgenic and wild-type (CC124) cells were cultured underphotoautotrophic conditions in nutrient replete [HS (Sueoka’s high saltmedium)] or in nitrogen-deprived (HS−N) media and visualized by laser scan-ning confocal microscopy. Pseudocolored representative images are shown(CrLPAAT2-mCherry, red; ER-tracker, green). (Scale bar, 1 µm.) (C) Magnifiedview of CrLPAAT2-mCherry association with lipid droplets under nitrogendeprivation. (Scale bar, 0.25 µm.)

observed that CrLPAAT2-mCherry largely colocalized with ERmembranes (Fig. 1B and SI Appendix, Fig. S4). The mCherry andthe ER-tracker signals showed positive association by Pearson’scorrelation coefficient, whereas the mCherry and the chlorophyllfluorescence signals displayed negative association (SI Appendix,Fig. S4), both in cells cultured in nutrient-replete medium and inthose under nitrogen deprivation.

Under nitrogen-starvation conditions, the strongest ER-tracker signal appeared to be associated with the ER and withthe periphery of LDs, which were detected with the nonpolarlipid fluorophore Nile Red (SI Appendix, Fig. S5). However,a fainter ER-tracker signal was also associated with LD cores(possibly due to the hydrophobic properties of the BODIPYFL dye) (Fig. 1B and SI Appendix, Fig. S4). Interestingly, inthese nitrogen-deprived cells, the CrLPAAT2-mCherry signal

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mostly overlapped the ER-tracker signal associated with theER and the periphery of LDs (Fig. 1C and SI Appendix, Fig.S4). Consistent with these observations, CrLPAAT2 has beenpreviously identified in proteomic analyses of isolated lipiddroplets from Chlamydomonas (26). These results suggested thatCrLPAAT2 might function in the eukaryotic pathway of glyc-erolipid assembly.

RNA Interference of CrLPAAT2 Mainly Affects TAG Accumula-tion Under Nitrogen Deprivation. To assess the in vivo role ofCrLPAAT2, expression of the corresponding gene was sup-pressed by RNA interference (RNAi) in transgenic strainsderived from CC124. As previously mentioned, CrLPAAT2 tran-script levels increase slightly under nitrogen deprivation in pho-toautotrophically grown cells (Fig. 2A). Several RNAi strainsshowed partial suppression of CrLPAAT2 expression and two(RNAi1 and RNAi2) were selected for further analyses. Thegrowth of these RNAi lines in nutrient-replete medium, underphotoautotrophic conditions, was very similar to that of thewild type (SI Appendix, Fig. S6). To evaluate nonpolar lipidaccumulation during nitrogen starvation, Chlamydomonas cellswere examined by fluorescence microscopy after staining withNile Red (19). LD formation, which normally increases sub-stantially in nitrogen-stressed cells, was reduced in the RNAistrains compared with the wild type (Fig. 2B). Likewise, TAG

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Fig. 2. RNA-mediated suppression of CrLPAAT2 expression in C. reinhardtii.(A) Transcript abundance of the indicated genes examined by semiquantita-tive reverse transcriptase (RT)-PCR in wild-type (CC124) and CrLPAAT2 RNAistrains. (B) Nonpolar lipid accumulation in the indicated strains, subject tonitrogen deprivation, examined by Nile Red staining. (Scale bar, 25 µm.)(C) Analysis of major lipids in CC124 and the CrLPAAT2 RNAi strains, culturedphotoautotrophically in nitrogen-depleted medium. Values shown are themean± SD of three independent experiments. Asterisks indicate significantdifferences (P < 0.05) in pairwise comparisons by a two-tailed Student’s t test.

content, determined as fatty acid methyl esters analyzed bygas chromatography–flame ionization detection (GC–FID), waslower in RNAi1 and RNAi2 (Fig. 2C and SI Appendix, Fig.S7). In contrast, the abundance of the major membrane glyc-erolipids was not affected in the RNAi strains (Fig. 2C). Interest-ingly, transgenic strains overexpressing the CrLPAAT2-mCherryfusion protein showed greater accumulation of nonpolar lipidsthan the parental CC124 strain (SI Appendix, Figs. S7 and S8).Overall, CrLPAAT2 appears to have a negligible role in cells cul-tured under nutrient-replete conditions, since its suppression didnot affect strain growth, but it is required for TAG biosynthesisunder nutrient deprivation.

CrLPAAT2 Prefers C16:0-CoA over C18:1-CoA as the Acyl Donor Sub-strate. Our results indicated that CrLPAAT2 localizes in theER and in the periphery of LDs and that it contributes toTAG accumulation under nitrogen deprivation. However, asalready discussed, Chlamydomonas TAGs mostly have C16 acylchains at the sn-2 position of their glycerol backbone (14, 27),instead of the sn-2-C18 acyl chains that are typical of TAGsassembled in the ER of land plants (9, 10). Thus, we decidedto test the acyltransferase activity of CrLPAAT2 to ascertainwhether it may have a substrate preference more similar to thatof prokaryotic (plastidial) LPAATs. Recombinant CrLPAAT2protein was produced by in vitro transcription/translation in acontinuous-exchange cell-free wheat-germ system and incubatedwith lysophosphatidic acid (sn-1-C18:1-lysoPA) and 14C-labeledpalmitoyl-CoA (∗C16:0-CoA) or oleoyl-CoA (∗C18:1-CoA). Theformation of labeled PA was examined by thin-layer chromatog-raphy (as shown for the oleoyl-CoA substrate in SI Appendix, Fig.S9) and quantified by liquid scintillation counting. Competitionexperiments were performed by adding an unlabeled acyl donor(palmitoyl-CoA or oleoyl-CoA) to the same reactions (Fig. 3A).

CrLPAAT2 showed minimal activity with sn-2-C16:0-lysoPAor sn-1-C18:1-MAG (monoacylglycerol) in comparison with sn-1-C18:1-lysoPA as the substrate (SI Appendix, Fig. S10). Withsn-1-C18:1-lysoPA and using either ∗C16:0-CoA (Fig. 3A, Left)or ∗C18:1-CoA (Fig. 3A, Right) as the labeled acyl donor, theaddition of unlabeled palmitoyl-CoA reduced the formation oflabeled PA to a greater extent than the addition of unlabeledoleoyl-CoA. This strongly indicated that CrLPAAT2, like plas-tidial LPAATs, prefers C16:0-CoA over C18:1-CoA as the acyldonor substrate. Moreover, examination of apparent enzymekinetics revealed that CrLPAAT2 exhibits a lower Km (Michaelisconstant) and a greater Vmax (maximum velocity) for C16:0-CoAthan for C18:1-CoA (SI Appendix, Fig. S11).

We chose to compare C16:0-CoA and C18:1-CoA as the acyldonor substrates for CrLPAAT2 because they correspond tothe main acyl chains incorporated into Chlamydomonas TAGsunder our experimental conditions (SI Appendix, Fig. S12A).However, besides substrate preference, in vivo enzyme activityis governed by the concentrations of available substrates. Thus,we also determined by liquid chromatography mass spectrome-try (LC-MS) the amounts of acyl-CoAs in C. reinhardtii grow-ing in nitrogen-depleted medium (Fig. 3B). C16:0-CoA was thesecond most abundant molecular species in the acyl-CoA pool(higher abundance than most C18-CoA species) which, togetherwith the CrLPAAT2 substrate preference, would support thepredominant incorporation of C16:0 at the sn-2 position of theglycerol backbone during TAG assembly in the ChlamydomonasER (SI Appendix, Fig. S12B). Even though C18:3-CoA was themajor species in the acyl-CoA pool (Fig. 3B), C18:3 was largelyexcluded from TAGs accumulated in nitrogen-deprived cells (SIAppendix, Fig. S12).

CrLPAAT2 Complements a Yeast LPAAT-Deleted Strain and ShiftsToward C16 the sn -2 Fatty Acid Composition of TAGs. To corrobo-rate CrLPAAT2 function in vivo, we expressed the recombinant

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Fig. 3. Recombinant CrLPAAT2 acyltransferase activity in vitro and relativeabundance of acyl-CoA species in vivo, in nitrogen-starved C. reinhardtii. (A)CrLPAAT2-dependent PA formation with 14C-labeled (indicated by an aster-isk) acyl-CoA donors (0.72 nmol) in the presence of increasing concentra-tions of unlabeled competitors. Representative results of three independentexperiments are shown. (B) Mole percent of acyl-CoA species in Chlamy-domonas cells determined by LC-MS. Values shown are the mean ± SD ofthree independent experiments.

protein in a double-knockout ale1∆-slc1∆ Saccharomyces cere-visiae strain. S. cerevisiae encodes one major LPAAT, termedAle1p (28), and one bacterial type LPAAT, named Slc1p (29).The double knockout of the corresponding genes causes lethal-ity (30), but the lethal phenotype was rescued by expression ofCrLPAAT2 from a multicopy plasmid (SI Appendix, Fig. S13). Asexpected, curing the double-knockout yeast strain of the plasmid,in the presence of 5-fluoroorotic acid, resulted again in lethality(SI Appendix, Fig. S13).

In yeast, TAG-containing LDs accumulate during station-ary phase (31), which resembles the accumulation of TAGsin Chlamydomonas under nitrogen starvation (14, 19). Sinceendogenous LPAAT activity is absent in the ale1∆-slc1∆ yeaststrain, the acyl chains esterified at the sn-2 position of TAGsin the CrLPAAT2-complemented strain are expected to reflectthe substrate preference of the Chlamydomonas enzyme. Consis-tent with the in vitro studies, we observed an increase in C16:0and C16:1 (and a decrease in C18:0) acyl chains at the sn-2 posi-tion of TAGs in the CrLPAAT2-complemented strain, relativeto the wild-type strain containing an empty vector (Fig. 4). Thus,CrLPAAT2 appears to favor, both in vitro and in vivo (albeit in aheterologous system), the esterification of C16 acyl chains at thesn-2 position of the glycerol backbone in the synthesis of precur-sors for TAG assembly.

DiscussionIn Chlamydomonas and related algae under nutrient depriva-tion, ∼70% of TAGs contain C16 acyl chains at the sn-2 positionof the glycerol backbone (14, 27), which led to the suggestionthat the plastidial pathway is the main source of precursorsfor TAG assembly. However, in plants, yeasts, and mammalsthe major assembly of storage TAGs seems to occur in theER (11–13). Moreover, an extensive number of TAG synthesisenzymes are predicted or verified to be located in the ER (11–13). Chlamydomonas appears to have duplicated sets of TAG

assembly enzymes targeted to the plastid and to the ER (3, 4),but the formation of large LDs within chloroplasts seems to berare, limited to certain mutant backgrounds, and/or under spe-cial environmental conditions (17, 18).

In wild-type C. reinhardtii, microscopy analyses revealed thatLDs formed under nitrogen deprivation appear to be primar-ily located in the cytosol (Fig. 1B), although in many casesin close association with the chloroplast envelope (18, 26, 32).These observations are consistent with the assembly of TAGspredominantly in ER membranes. Thus, the hypothesis that pre-cursor molecules are mainly generated by the plastidial path-way would require a strong flux of glycerolipids (presumablyPA and/or diacylglycerol) from the chloroplast to the ER (SIAppendix, Fig. S14A). However, to our knowledge, the evidencefor glycerolipid trafficking from plastids to the ER is limited(33), although the reverse flux is well established by the anal-ysis of trigalactosyldiacylglycerol (TGD) transporters in plants(34). Instead, our results suggest that, in C. reinhardtii and relatedmicroalgae under nutritional stress, most TAG precursors aresynthesized in the ER (Fig. 5). However, the substrate specificityof the chlorophyte-specific LPAAT involved in this pathway,CrLPAAT2, determines the preferential esterification of C16acyl chains at the sn-2 position of the glycerol backbone and thesynthesis of “prokaryotic” glycerolipid precursors in the ER.

Several lines of evidence support this hypothesis. The pref-erence of C16:0-CoA over C18:1-CoA as the substrate is muchhigher for plastidial CrLPAAT1 (15) than for the ER-locatedCrLPAAT2 (SI Appendix, Fig. S11). In the RNAi strains, whereCrLPAAT2 expression has been partly suppressed, the existenceof a prevalent plastidial pathway would then be expected to con-tribute a greater proportion of TAG precursors than in the wild-type strain, resulting in an increase in the C16 acyl chains esteri-fied at the sn-2 position of the accumulated TAGs (SI Appendix,Fig. S14B). However, this was not observed (SI Appendix, Fig.S12B). Instead, in agreement with a model where most TAGprecursors are synthesized by the ER-located CrLPAAT2 (Fig.5), with only a minor contribution from the plastidial pathway,RNA-mediated CrLPAAT2 suppression reduced TAG accumu-lation (Fig. 2 and SI Appendix, Figs. S7 and S8) without alter-ing the ratio of acyl chains esterified at the sn-2 position (SIAppendix, Fig. S12B). This is expected for an overall reductionin enzymatic activity (due to the RNAi-mediated reduction inCrLPAAT2 mRNA abundance), whereas the substrate specificityof CrLPAAT2 would not be affected.

Our data are also consistent with proposed intracellularglycerolipid fluxes. 14C-acetate pulse-chase experiments, and theexistence of a TGD2 orthologue, suggested that, in Chlamy-domonas, glycerolipid precursors (possibly PA) could be trans-ferred from the ER to the chloroplast envelope for the synthesis

Fig. 4. Mole percent of fatty acid methyl esters (FAMEs) derived from thesn-2 position of TAGs, in wild-type S. cerevisiae containing an empty vector(ALE1-SLC1-EV) and in a LPAAT-deleted strain complemented with CrLPAAT2(ale1∆-slc1∆-CrLPAAT2). Values shown are the mean ± SD of three inde-pendent experiments. Asterisks indicate significant differences (P < 0.05) inpairwise comparisons by a two-tailed Student’s t test.

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Fig. 5. Proposed pathway for TAG assembly in nitrogen-deprived C. rein-hardtii under photoautotrophic conditions. See text for details. CrLPAAT1/2,lysophosphatidic acid acyltransferase; DGTT1/2/3, diacylglycerol acyltrans-ferase; FAT, fatty acyl-ACP thioesterase; GPAT, glycerol-3-phosphate acyl-transferase; PAP, phosphatidic acid phosphatase; TGD2, trigalactosyldiacyl-glycerol 2.

of thylakoid lipids (35). However, thylakoid lipids in Chlamy-domonas lack C18 fatty acids at the sn-2 position (3, 35), imply-ing that ER-synthesized precursors must have C16 acyl chainsesterified at this position. Thus, CrLPAAT2 could generate sn-2-C16-PA in the ER for assembly into TAGs and/or for trans-fer to the plastid, for assembly into monogalactosyldiacylglyc-erol (MGDG) (Fig. 5). In turn, since newly synthesized MGDGappears to be partly degraded under nitrogen deprivation (36),the corresponding acyl chains may return to the ER or to LDmembranes for TAG assembly (26).

A meaningful role of CrLPAAT2 in TAG biosynthesis inChlamydomonas, under nutrient deprivation, is also supportedby the enhanced accumulation of nonpolar lipids in the trans-genic strains overexpressing CrLPAAT2-mCherry (SI Appendix,Figs. S7 and S8). DGTT1, a Chlamydomonas DGAT that cat-alyzes the last step in TAG synthesis, was found to localizeprimarily to the ER (and also somewhat to the chloroplastenvelope) and to prefer sn-2-C16-DAG over sn-2-C18-DAGas the substrate (37). Barring substantial glycerolipid traf-ficking from plastid to the ER, CrLPAAT2 could generatedirectly in ER membranes the prokaryotic precursor preferredfor DGTT1-mediated TAG assembly. However, ER membraneglycerolipids, such as diacylglycerol-N,N,N-trimethylhomoserine(DGTS), phosphatidylethanolamine (PE), or phosphatidylinos-itol (PI), mostly have C18:3 esterified at the sn-2 positionin Chlamydomonas (3). Thus, other acyltransferases, possiblyacyl-CoA:lysophospholipid acyltransferase-like enzymes (SIAppendix, Fig. S2) (38), may synthesize the precursors involvedin ER membrane glycerolipid assembly. We propose that, inChlamydomonas and related microalgae, CrLPAAT2 may syn-thesize prokaryotic glycerolipid precursors mainly for assem-bly into TAGs and/or transfer to the plastid whereas otheracyltransferase(s) may synthesize “eukaryotic” glycerolipid pre-cursors mainly for assembly into DGTS/PE/PI. This hypothe-sis implies discrimination between intermediates in the TAGand DGTS/PE/PI biosynthesis pathways (possibly through sub-strate channeling and/or substrate preference among consecu-tive enzymes within each pathway) and/or subcompartmentationof the two assembly pathways within the ER of microalgae, asalready suggested in other eukaryotes (39, 40).

In summary, our data strongly indicate that CrLPAAT2, achlorophyte-specific acyltransferase, is localized to the ER but,unlike canonical eukaryotic LPAATs, prefers C16:0-CoA as

the substrate. CrLPAAT2 appears to contribute to TAG accu-mulation under nitrogen deprivation by generating prokary-otic glycerolipid intermediates for TAG assembly. However, agreater understanding of lipid metabolism in Chlamydomonas,particularly as it relates to TAG and membrane glycerolipidassembly and to intracellular lipid trafficking, will require char-acterization of additional acyltransferases. From a practical per-spective, an appreciation of the differences in lipid metabolismbetween microalgae and other eukaryotes may be necessary forthe biotechnological development of algae as sustainable feed-stocks for biofuel and biomaterial production.

Materials and MethodsStrains and Culture Conditions. C. reinhardtii CC124 and derived transgenicstrains were used in all reported experiments. Cells were grown photoau-totrophically in nutrient-replete (HS) or in nitrogen-depleted (HS−N) media(41), as previously described (5, 19). Yeast ale1∆ and slc1∆ mutants weresupplied by W.R.R. and grown in synthetic growth medium, as previouslyreported (42).

Phylogenetic and Bioinformatic Analyses. Cre17.g738350 sequences wereretrieved from the Chlamydomonas genome v5.5 (16). BLASTP searchesagainst GenBank, Phytozome v11.0, and UniProt identified related LPAATsequences in diverse eukaryotes. Various bioinformatic tools were used tobuild phylogenetic trees and to obtain CrLPAAT2 domain information (43–47), as detailed in SI Appendix, SI Materials and Methods.

Construction of a CrLPAAT2-mCherry Fusion, Generation of Transgenic Strains,and Fluorescence Microscopy. Construction of the CrLPAAT2-mCherry trans-gene is described in detail in SI Appendix, SI Materials and Methods. Thistransgene was transformed into CC124 by electroporation and the subcellu-lar localization of fluorescent proteins examined by laser scanning confocalmicroscopy, as previously reported (5, 24). Cells were also stained with Nile Red(Sigma), as described in ref. 19, and/or with ER-Tracker Green (ThermoFisherScientific), by incubating iodine-treated cells with 2 µM of the dye.

Generation of CrLPAAT2 RNAi Transgenic Strains. Chlamydomonas strainscontaining an inverted repeat transgene homologous to Cre17.g738350were obtained as described in SI Appendix, SI Materials and Methods, fol-lowing established protocols (5, 48).

Semiquantitative RT-PCR. The expression of CrLPAAT2 was examined bysemiquantitative PCR methods, as described previously (5, 19). Primers andPCR conditions are detailed in SI Appendix, SI Materials and Methods.

CrLPAAT2 Acyltransferase Activity and Competition Assays. RecombinantCrLPAAT2 was produced by in vitro transcription/translation in a cell-freewheat-germ system (Biotechrabbit), in accordance with the manufacturer’sprotocol. The 14C-palmitoyl-CoA (60 µCi/µmol; PerkinElmer) or 14C-oleoyl-CoA (60 µCi/µmol; PerkinElmer) was incubated with oleoyl-lysophosphatidicacid (Avanti Polar Lipids) and recombinant CrLPAAT2 to examine the forma-tion of 14C-PA, as described in previous reports (8). Details of the proto-col and of the competition assays with unlabeled palmitoyl-CoA (Sigma) oroleoyl-CoA (Sigma) are provided in SI Appendix, SI Materials and Methods.

Lipid Analyses. Total lipids from C. reinhardtii or S. cerevisiae were analyzedas described in refs. 5, 19, and 49. To identify fatty acids esterified at thesn-2 position of the glycerol backbone, TAGs were digested with a Rhizopusoryzae TAG lipase (62305; Sigma), as outlined in SI Appendix, SI Materialsand Methods.

Complementation of the S. cerevisiae ale1∆-slc1∆ Mutant Strain. TheCre17.g738350 coding sequence was cloned into the pYES2.1 TOPO vector(ThermoFisher Scientific), following the manufacturer’s protocol, and trans-formed into yeast. The procedure, strain crossing, and spore analyses areexplained in detail in SI Appendix, SI Materials and Methods; 5-fluorooroticacid (5-FOA), at 5 mg/mL, was used to cure the plasmid from the yeast strains.

ACKNOWLEDGMENTS. We thank Rebecca Cahoon and Rebecca Ros-ton for help with liquid chromatography-mass spectrometry and gaschromatography-flame ionization detection. This work was supported inpart by a grant from the National Science Foundation (to H.C.). We alsoacknowledge the support of the EPSCoR (Established Program to StimulateCompetitive Research) program (EPS-1004094 to W.R.R., E.B.C., and H.C.).

1656 | www.pnas.org/cgi/doi/10.1073/pnas.1715922115 Kim et al.

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