Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas

Yeongho 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 review September 9, 2017) Understanding the unique features of triacylglycerol (TAG) metab- transferase (PDAT), are also consistent with TAG assembly tak- olism in microalgae may be necessary to realize the full poten- ing place in the ER (11–13). tial of these organisms for biofuel and biomaterial production. In In contrast, in C. reinhardtii, TAGs accumulated under nitro- the unicellular green alga Chlamydomonas reinhardtii a chloro- gen deprivation mostly have C16 at their sn-2 position and it has plastic (prokaryotic) pathway has been proposed to play a major been hypothesized that the plastidial pathway plays a major role in role in TAG precursor biosynthesis. However, as reported here, C. TAG synthesis (4, 14, 15). Since a canonical, ER-targeted LPAAT reinhardtii contains a chlorophyte-specific lysophosphatidic acid was not identified in the C. reinhardtii genome (16), the plas- acyltransferase, CrLPAAT2, that localizes to endoplasmic reticulum tidial chlorophyte-specific (Cr)LPAAT1 has been suggested to (ER) membranes. Unlike canonical, ER-located LPAATs, CrLPAAT2 participate actively in generating precursors for TAG accumula- prefers palmitoyl-CoA over oleoyl-CoA as the acyl donor sub- tion (15). Moreover, light and electron microscopy revealed LDs strate. RNA-mediated suppression of CrLPAAT2 indicated that the in both the cytosol and the chloroplast of nutrient-starved C. rein- enzyme is required for TAG accumulation under nitrogen depri- hardtii, although plastid-located large LDs were observed only in vation. Our findings suggest that Chlamydomonas has a distinct starchless mutants deprived of nitrogen under mixotrophic con- glycerolipid assembly pathway that relies on CrLPAAT2 to gener- ditions or in wild-type strains under special environmental con- ate prokaryotic-like TAG precursors in the ER. ditions such as saturating light (17, 18). These findings, together with the predicted subcellular location of major enzymes of TAG LPAAT | lipid droplets | triacylglycerol metabolism | algae | biofuels biosynthesis (3, 4), support the involvement of both the prokary- otic and eukaryotic pathways in algal TAG assembly, but their riacylglycerol (TAG) is a major storage lipid in most eukary- contributions may vary depending on cultivation conditions and Totes and a precursor for biodiesel production (1, 2). Some strain genotype. Additionally, LPAATs have not been character- microalgae have recently gained attention because they can accu- ized in detail in microalgae and it is not certain that the plant mulate large amounts of TAGs and potentially serve as feedstock paradigm regarding LPAAT substrate specificity in the two path- for biofuel production (2–4). However, despite current advances, ways applies universally to algal species. our understanding of algal lipid metabolism is still fairly lim- Here we demonstrate that C. reinhardtii contains a unique ited and generally based on insights from land plants, even for LPAAT, encoded by Cre17.g738350 and termed CrLPAAT2, the well-studied model system Chlamydomonas reinhardtii (3, which seems to be restricted to the chlorophytes. CrLPAAT2 is 4). However, algal metabolism appears to have some distinct localized to the ER but, like prokaryotic acyltransferases, prefers features (4, 5) whose understanding may be required for the C16:0-CoA over C18:1-CoA as the substrate. RNA-mediated biotechnological improvement of algal strains. During the biogenesis of complex lipids in plants, fatty acids Significance synthesized de novo in the chloroplast can be assembled into glycerolipids by the prokaryotic (plastidial) pathway or they The acyl chain composition of Chlamydomonas triacylglyc- can be exported to the endoplasmic reticulum (ER), entering erols (TAGs) suggests that they are assembled from prokary- the eukaryotic pathway of glycerolipid assembly. Glycerolipids otic precursors, proposed to be synthesized in the chloroplast. synthesized by the prokaryotic pathway carry a 16-carbon acyl However, in most eukaryotes, the endoplasmic reticulum (ER) chain at the sn-2 position of the glycerol backbone, whereas appears to be the main organelle for storage TAG biosyn- glycerolipids assembled by the eukaryotic pathway contain an thesis. Interestingly, Chlamydomonas reinhardtii has a dis- 18-carbon acyl chain at the same position (6). This distinc- tinct lysophosphatidic acid acyltransferase that localizes to the tion is caused by differences in the substrate specificity of ER but resembles prokaryotic lysophosphatidic acid acyltrans- lysophosphatidic acid acyltransferases (LPAATs). Chloroplast- ferases (LPAATs) in its substrate preference. Thus, Chlamy- localized LPAATs mainly use palmitoyl-ACP (C16:0-ACP) domonas and related green algae, unlike land plants, can as the acyl donor to generate sn-2-C16:0-phosphatidic acid synthesize “prokaryotic” acyl-lipids in the ER, with intriguing (PA) (7, 8) while those in the ER prefer using oleoyl- implications for biotechnological applications. CoA (C18:1-CoA) to synthesize sn-2-C18:1-PA (9, 10). These observations have been recapitulated in numerous land plants Author contributions: Y.K. and H.C. designed research; Y.K. and E.L.T. performed (7–11). research; Y.K., E.L.T., W.R.R., E.B.C., and H.C. analyzed data; and Y.K. and H.C. wrote the In plant seeds, the assembly of storage TAGs occurs in the paper. ER, having C18 esterified at the sn-2 position of their glycerol The authors declare no conflict of interest. backbone (11). Moreover, in plants, mammals, and fungi, micro- This article is a PNAS Direct Submission. scopic observations of TAG-containing lipid droplets (LDs) Published under the PNAS license. and the subcellular location of major enzymes involved in the 1 To whom correspondence should be addressed. Email: [email protected]. final step of TAG synthesis, such as acyl-CoA:diacylglycerol This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. acyltransferases (DGATs) and phospholipid:diacylglycerol acyl- 1073/pnas.1715922115/-/DCSupplemental.

1652–1657 | PNAS | February 13, 2018 | vol. 115 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1715922115 Downloaded by guest on September 28, 2021 Downloaded by guest on September 28, 2021 i tal. et Kim by Indeed, we (25), S1). Glibenclamide) Table FL (BODIPY Appendix, dye (SI ER-tracker an pathway using secretory of the absence to the verify channel. to mCherry the control, in negative signal a background any as used was 1B CC124 (Fig. imaging cytosol the in mainly of localize to into found the was electroporation CrLPAAT2-mCherry of by control at introduced the frame loca- then under in subcellular transgene, fused This was its sequence 5 examined coding the CrLPAAT2 we The function tion. CrLPAAT2 Reticulum. ing Endoplasmic the to Localizes CrLPAAT2 phy- and as eukaryotic well LPAATs. well-characterized as prokaryotic from structurally divergent LPAATs, a algal logenetically represent of to group appears CrLPAAT2 distinctive Thus, LPAATs. from plastidial eukaryotic or distinguished (LPAT2-5s), clearly (LPCATs/LPEATs), LPAATs acyltransferases was acyl-CoA:lysophospholipid eukaryotic clade containing this those and chloro- exclusively eukaryotes proteins; containing clade phyte assorted well-supported CrLPAAT2 a from Interestingly, to S2). belongs those Fig. Appendix, (SI and eubacteria LPAAT- and sequences available algal with performed related were analyses phylogenetic plastidial from domains LPAATs. combining eukaryotic and structure, chimeric a have to from AtLPAT2 1A simi- as (Fig. such structure LPAATs a thaliana eukaryotic TMs, bidopsis of predicted that (Fig. to 15) two lar (8, contains LPAATs CrLPAAT2 sim- chloroplast of fairly of is region CrLPAAT2 InterproScan central 1A of by the half sites to N-terminal ilar catalytic the of that and revealed model) (transmembrane peptide Markov TMHMM transit by hidden chloroplast (TMs) domains a transmembrane have 1 not (Fig. Inter- does suggested algorithms the CrLPAAT2 PredAlgo that by and ChloroP identified program. proScan (PANTHER10434) LPAAT/ a domain contains AGPAT LPAATs, Chloro- conventional a like to Cre17.g738350, Belongs Clade. and Features phyte-Specific Structural Unique Has CrLPAAT2 deprivation. its nutritional investigate under metabolism to acyl-lipid decided in of we role the features enzyme, distinctive CrLPAAT2 under mod- some predicted given this below) the expression, Despite gene (see in conditions. change slightly photoautotrophic est con- increased in mixotrophic stress but in same 21) deprivation (20, nitrogen ditions under stable abun- atively transcript to The of (14). appeared biosynthesis dance increased synthesis TAG an toward acid despite flux conditions, carbon fatty these novo under pre- down-regulated de be genes in several involved Moreover, sumably (20–22). unexpect- metabolism acyl-lipid somewhat in nitrogen-depleted However, that 19). revealed Chlamydomonas approaches (2, systems-level microalgae edly, stress nutritional many by triggered in is accumulation TAG Substantial Results prokaryotic- generate ER. to the in CrLPAAT2 species on TAG like rely to appears photoau- algae) in required Thus, deprivation is conditions. nitrogen enzyme totrophic under the accumulation that TAG indicated for CrLPAAT2 of silencing iifrai nlsssgetdta rPA2i targeted is CrLPAAT2 that suggested analyses Bioinformatic CrLPAAT2, of origin evolutionary the into insight gain To ybt iecl n immunofluorescence and live-cell both by Chlamydomonas, and 0 n ftemhryfloecn rti eune(24). sequence protein fluorescent mCherry the of end A .I otat h -emnlhalf C-terminal the contrast, In S1). Fig. Appendix, SI and and noigCLAT,rmie rel- remained CrLPAAT2, encoding Cre17.g738350, .Ide,CLAT seems CrLPAAT2 Indeed, S1). Fig. Appendix , SI .Hwvr nlssof analyses However, S1). Table Appendix, SI hwdu-euaino e ee involved genes few of up-regulation showed n A1from LAT1 and and h rdce mn cdsqec of sequence acid amino predicted The .Nontransgenic S3). Fig. Appendix, SI Chlamydomonas innhsdouglasii Limnanthes .reinhardtii C. PsaD adrltdgreen related (and obgnassess- begin To rmtr was promoter, CC124. 9 23) (9, Ara- hs irgndpie el,teCLAT-Cer signal CrLPAAT2-mCherry the cells, nitrogen-deprived these psil u otehdohbcpoete fteBODIPY the of 1B cores properties LD (Fig. hydrophobic with However, dye) the associated S5). FL to also Fig. was due nonpolar Appendix , signal (possibly the (SI ER-tracker with fainter Red detected with a and Nile were ER which fluorophore the LDs, lipid with of associated periphery be the to appeared signal tracker in and deprivation. medium nutrient-replete nitrogen in under cultured those cells in both Appendix, S4), (SI Fig. association negative displayed chlorophyll signals the fluorescence and Pearson’s mCherry the by whereas association coefficient, positive correlation showed signals ER-tracker the ER 1B with (Fig. colocalized membranes largely CrLPAAT2-mCherry that observed nitrogen under droplets lipid 0.25 bar, (Scale with deprivation. association CrLPAAT2-mCherry of view 1 bar, shown (Scale are green). salt ER-tracker, images red; (CrLPAAT2-mCherry, representative high Pseudocolored microscopy. (Sueoka’s confocal [HS ning replete (HS nitrogen-deprived in nutrient under or medium)] cultured in were cells conditions (CC124) photoautotrophic wild-type CrLPAAT2-mCherry and the Transgenic transmem- of protein. and localization fusion Subcellular (black), (B) domain (gray). catalytic domains LPAAT chloro- brane (red), domains: peptide key transit indicating diagrams plast protein Schematic (A) CrLPAAT2. 1. Fig. C B A ne irgnsavto odtos h togs ER- strongest the conditions, nitrogen-starvation Under ceai igaso PAsadsbellrlclzto of localization subcellular and LPAATs of diagrams Schematic PNAS and and | µm.) i.S4 Fig. Appendix, SI .Temhryand mCherry The S4). Fig. Appendix, SI eray1,2018 13, February )mdaadvsaie ylsrscan- laser by visualized and media −N) | o.115 vol. .Itrsigy in Interestingly, ). . (C µm.) | o 7 no. Magnified ) | 1653

PLANT BIOLOGY mostly overlapped the ER-tracker signal associated with the content, determined as fatty acid methyl esters analyzed by ER and the periphery of LDs (Fig. 1C and SI Appendix, Fig. gas chromatography–flame ionization detection (GC–FID), was S4). Consistent with these observations, CrLPAAT2 has been lower in RNAi1 and RNAi2 (Fig. 2C and SI Appendix, Fig. previously identified in proteomic analyses of isolated lipid S7). In contrast, the abundance of the major membrane glyc- droplets from Chlamydomonas (26). These results suggested that erolipids was not affected in the RNAi strains (Fig. 2C). Interest- CrLPAAT2 might function in the eukaryotic pathway of glyc- ingly, transgenic strains overexpressing the CrLPAAT2-mCherry erolipid assembly. fusion protein showed greater accumulation of nonpolar lipids than the parental CC124 strain (SI Appendix, Figs. S7 and S8). RNA Interference of CrLPAAT2 Mainly Affects TAG Accumula- Overall, CrLPAAT2 appears to have a negligible role in cells cul- tion Under Nitrogen Deprivation. To assess the in vivo role of tured under nutrient-replete conditions, since its suppression did CrLPAAT2, expression of the corresponding gene was sup- not affect strain growth, but it is required for TAG biosynthesis pressed by RNA interference (RNAi) in transgenic strains under nutrient deprivation. derived from CC124. As previously mentioned, CrLPAAT2 tran- script levels increase slightly under nitrogen deprivation in pho- CrLPAAT2 Prefers C16:0-CoA over C18:1-CoA as the Acyl Donor Sub- toautotrophically grown cells (Fig. 2A). Several RNAi strains strate. Our results indicated that CrLPAAT2 localizes in the showed partial suppression of CrLPAAT2 expression and two ER and in the periphery of LDs and that it contributes to (RNAi1 and RNAi2) were selected for further analyses. The TAG accumulation under nitrogen deprivation. However, as growth of these RNAi lines in nutrient-replete medium, under already discussed, Chlamydomonas TAGs mostly have C16 acyl photoautotrophic conditions, was very similar to that of the chains at the sn-2 position of their glycerol backbone (14, 27), wild type (SI Appendix, Fig. S6). To evaluate nonpolar lipid instead of the sn-2-C18 acyl chains that are typical of TAGs accumulation during nitrogen starvation, Chlamydomonas cells assembled in the ER of land plants (9, 10). Thus, we decided were examined by fluorescence microscopy after staining with to test the acyltransferase activity of CrLPAAT2 to ascertain Nile Red (19). LD formation, which normally increases sub- whether it may have a substrate preference more similar to that stantially in nitrogen-stressed cells, was reduced in the RNAi of prokaryotic (plastidial) LPAATs. Recombinant CrLPAAT2 strains compared with the wild type (Fig. 2B). Likewise, TAG protein was produced by in vitro transcription/translation in a continuous-exchange cell-free wheat-germ system and incubated with lysophosphatidic acid (sn-1-C18:1-lysoPA) and 14C-labeled A palmitoyl-CoA (∗C16:0-CoA) or oleoyl-CoA (∗C18:1-CoA). The formation 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. Competition experiments 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-lysoPA or sn-1-C18:1-MAG (monoacylglycerol) in comparison with sn- 1-C18:1-lysoPA as the substrate (SI Appendix, Fig. S10). With B sn-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, the addition of unlabeled palmitoyl-CoA reduced the formation of labeled PA to a greater extent than the addition of unlabeled oleoyl-CoA. This strongly indicated that CrLPAAT2, like plas- tidial LPAATs, prefers C16:0-CoA over C18:1-CoA as the acyl donor substrate. Moreover, examination of apparent enzyme kinetics revealed that CrLPAAT2 exhibits a lower Km (Michaelis constant) and a greater Vmax (maximum velocity) for C16:0-CoA than for C18:1-CoA (SI Appendix, Fig. S11). We chose to compare C16:0-CoA and C18:1-CoA as the acyl donor substrates for CrLPAAT2 because they correspond to C the main acyl chains incorporated into Chlamydomonas TAGs under our experimental conditions (SI Appendix, Fig. S12A). However, besides substrate preference, in vivo enzyme activity is 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 the second most abundant molecular species in the acyl-CoA pool (higher abundance than most C18-CoA species) which, together with the CrLPAAT2 substrate preference, would support the predominant incorporation of C16:0 at the sn-2 position of the glycerol backbone during TAG assembly in the Chlamydomonas Fig. 2. RNA-mediated suppression of CrLPAAT2 expression in C. reinhardtii. ER (SI Appendix, Fig. S12B). Even though C18:3-CoA was the (A) Transcript abundance of the indicated genes examined by semiquantita- major species in the acyl-CoA pool (Fig. 3B), C18:3 was largely tive reverse transcriptase (RT)-PCR in wild-type (CC124) and CrLPAAT2 RNAi excluded from TAGs accumulated in nitrogen-deprived cells (SI strains. (B) Nonpolar lipid accumulation in the indicated strains, subject to nitrogen deprivation, examined by Nile Red staining. (Scale bar, 25 µm.) Appendix, Fig. S12). (C) Analysis of major lipids in CC124 and the CrLPAAT2 RNAi strains, cultured photoautotrophically in nitrogen-depleted medium. Values shown are the CrLPAAT2 Complements a Yeast LPAAT-Deleted Strain and Shifts mean ± SD of three independent experiments. Asterisks indicate significant Toward C16 the sn -2 Fatty Acid Composition of TAGs. To corrobo- differences (P < 0.05) in pairwise comparisons by a two-tailed Student’s t test. rate CrLPAAT2 function in vivo, we expressed the recombinant

1654 | www.pnas.org/cgi/doi/10.1073/pnas.1715922115 Kim et al. Downloaded by guest on September 28, 2021 Downloaded by guest on September 28, 2021 nye r rdce rvrfidt elctdi h R(11– ER the the in in located be occur to synthesis verified to TAG 13). or of seems predicted number are TAGs extensive mammals precursors enzymes an storage and of Moreover, yeasts, of (11–13). source ER plants, assembly main in suggestion major the However, the the is assembly. to led TAG pathway which for plastidial 27), the (14, backbone that glycerol the of tion, In Discussion precur- of synthesis assembly. the TAG in for backbone the sors glycerol at the chains of a acyl position in C16 sn-2 (albeit of vivo esterification in the and system), Thus, vitro heterologous in 4). both (Fig. favor, vector to empty appears relative an CrLPAAT2 containing strain, strain CrLPAAT2-complemented wild-type the the C16:0 the at to in in chains increase TAGs acyl C18:0) an of in observed tion decrease we a studies, (and C16:1 vitro and in the with reflect tent the to of expected preference the substrate are the strain at CrLPAAT2-complemented esterified the chains in the TAGs acyl in the of absent strain, is accumulation activity the LPAAT endogenous resembles which in (31), phase ary lethality in again S13). resulted Fig. acid, Appendix, 5-fluoroorotic (SI of presence plasmid, the the of of in strain expression yeast As double-knockout by S13). the Fig. curing rescued , Appendix expected, (SI was plasmid phenotype multicopy a lethal lethal- from CrLPAAT2 causes the (29). genes but Slc1p corresponding (30), named the ity LPAAT, of type knockout double bacterial The one and (28), Ale1p visiae double-knockout a in protein in species experiments. acyl-CoA independent of three percent Mole concentra- (B) domonas increasing shown. of independent are three presence of experiments results the Representative in competitors. unlabeled nmol) of tions (0.72 donors acyl-CoA isk) rPA2dpnetP omto with nitrogen-starved formation in PA CrLPAAT2-dependent vivo, in species acyl-CoA of abundance 3. Fig. i tal. et Kim B A nyat A-otiigLsacmlt uigstation- during accumulate LDs TAG-containing yeast, In Chlamydomonas Chlamydomonas Chlamydomonas 0 fTG oti 1 clcan tthe at chains acyl C16 contain TAGs of ∼70% strain. eobnn rPA2aytaseaeatvt nvtoadrelative and vitro in activity acyltransferase CrLPAAT2 Recombinant el eemndb CM.Vle hw r h mean the are shown Values LC-MS. by determined cells .cerevisiae S. n eae la ne uretdepriva- nutrient under algae related and ne irgnsavto 1,1) Since 19). (14, starvation nitrogen under per ohv ulctdst fTAG of sets duplicated have to appears noe n ao PA,termed LPAAT, major one encodes ale1∆ Chlamydomonas 14 -aee idctdb naster- an by (indicated C-labeled -slc1∆ 2psto fTAGs of position sn-2 acaoye cere- Saccharomyces ale1∆ nye Consis- enzyme. (A) reinhardtii. C. 2position sn-2 -slc1∆ 2posi- sn-2 ± Chlamy- yeast Dof SD erdfo h Rt h hools neoefrtesynthesis the for envelope chloroplast trans- the in to be ER that, could the PA) from suggested ferred (possibly orthologue, precursors glycerolipid TGD2 domonas, a of existence fluxes. glycerolipid affected. be in not reduction would CrLPAAT2 RNAi-mediated of reduction the overall to an the (due CrLPAAT2 for at activity expected esterified enzymatic is in chains This S12B). acyl Fig. of Appendix, ratio the and ing pathway, 2 plastidial (Fig. the lation from contribution TAG minor (Fig. most RNA-mediated a CrLPAAT2 where only ER-located with model the Fig. 5), a by Appendix, synthesized with ( SI are agreement observed precursors in not Instead, was this S12B). However, esteri- chains S14B). acyl Fig. C16 the the in at wild- increase fied the an in in than resulting precursors con- strain, TAG to type of expected proportion be greater then a would tribute pathway plastidial prevalent where a strains, of ER-located RNAi the the In for S11). CrLPAAT2 than Fig. much Appendix, (15) is (SI CrLPAAT1 substrate CrLPAAT2 the plastidial as for C18:1-CoA higher over C16:0-CoA of erence ER. the in precursors glycerolipid C16 “prokaryotic” of of pathway, synthesis the esterification this at chains preferential in acyl the involved determines LPAAT CrLPAAT2, specificity chlorophyte-specific substrate are the the However, precursors 5). of (Fig. TAG ER most the in stress, synthesized nutritional anal- under the plants in microalgae that, by in suggest limited results established transporters our is Instead, well (TGD) (34). ER is trigalactosyldiacylglycerol the flux of reverse to ysis the plastids (SI although from ER (33), trafficking the glycerolipid (presumably to for glycerolipids chloroplast of S14 the Fig. from flux path- Appendix, diacylglycerol) plastidial strong and/or the a PA by require generated would mainly way TAGs are pre- cases of that molecules hypothesis many assembly the 32). cursor Thus, the in 26, membranes. with ER (18, although in consistent envelope predominantly 1B), are chloroplast observations the (Fig. with These primar- cytosol association be the close to in in appear deprivation located nitrogen ily under formed LDs spe- under 18). be and/or (17, to backgrounds, conditions 4), seems environmental mutant (3, chloroplasts cial certain within ER to LDs the limited large to rare, of and formation plastid the the but to targeted enzymes assembly ariecmaiosb w-aldStudent’s two-tailed a by ( comparisons differences pairwise significant indicate Asterisks mean experiments. the pendent are shown CrLPAAT2 Values with (ale1∆-slc1-CrLPAAT2). complemented strain LPAAT-deleted a in and wild-type (ALE1-SLC1-EV) in TAGs, of position sn-2 4. Fig. u aaaeas ossetwt rpsdintracellular proposed with consistent also are data Our pref- The hypothesis. this support evidence of lines Several wild-type In oepreto at cdmty ses(AE)drvdfo the from derived (FAMEs) esters methyl acid fatty of percent Mole , Appendix (SI TAGs accumulated the of position sn-2 RAaudne,weestesbtaespecificity substrate the whereas abundance), mRNA xrsinhsbe atysprse,teexistence the suppressed, partly been has expression irsoyaaye eeldthat revealed analyses microscopy reinhardtii, C. CrLPAAT2 PNAS 2psto ftegyeo akoeadthe and backbone glycerol the of position sn-2 .Hwvr oorkolde h evidence the knowledge, our to However, A). ihu alter- without S8) and S7 Figs. Appendix, SI 14 -ctt us-hs xeiet,adthe and experiments, pulse-chase C-acetate | eray1,2018 13, February upeso eue A accumu- TAG reduced suppression .cerevisiae S. .reinhardtii C. t otiiga mt vector empty an containing | test. o.115 vol. ± 2psto (SI position sn-2 Do he inde- three of SD | o 7 no. n related and P < Chlamy- .5 in 0.05) | 1655

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

Materials and Methods Strains and Culture Conditions. C. reinhardtii CC124 and derived transgenic strains 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 were supplied by W.R.R. and grown in synthetic growth medium, as previously Fig. 5. Proposed pathway for TAG assembly in nitrogen-deprived C. rein- reported (42). hardtii under photoautotrophic conditions. See text for details. CrLPAAT1/2, lysophosphatidic acid acyltransferase; DGTT1/2/3, diacylglycerol acyltrans- Phylogenetic and Bioinformatic Analyses. Cre17.g738350 sequences were ferase; FAT, fatty acyl-ACP thioesterase; GPAT, glycerol-3-phosphate acyl- retrieved from the Chlamydomonas genome v5.5 (16). BLASTP searches transferase; PAP, phosphatidic acid phosphatase; TGD2, trigalactosyldiacyl- against GenBank, Phytozome v11.0, and UniProt identified related LPAAT glycerol 2. sequences in diverse eukaryotes. Various bioinformatic tools were used to build phylogenetic trees and to obtain CrLPAAT2 domain information (43– 47), as detailed in SI Appendix, SI Materials and Methods. of thylakoid lipids (35). However, thylakoid lipids in Chlamy- domonas lack C18 fatty acids at the sn-2 position (3, 35), imply- Construction of a CrLPAAT2-mCherry Fusion, Generation of Transgenic Strains, ing that ER-synthesized precursors must have C16 acyl chains and Fluorescence Microscopy. Construction of the CrLPAAT2-mCherry trans- esterified at this position. Thus, CrLPAAT2 could generate sn- gene is described in detail in SI Appendix, SI Materials and Methods. This 2-C16-PA in the ER for assembly into TAGs and/or for trans- transgene was transformed into CC124 by electroporation and the subcellu- lar localization of fluorescent proteins examined by laser scanning confocal fer to the plastid, for assembly into monogalactosyldiacylglyc- microscopy, as previously reported (5, 24). Cells were also stained with Nile Red erol (MGDG) (Fig. 5). In turn, since newly synthesized MGDG (Sigma), as described in ref. 19, and/or with ER-Tracker Green (ThermoFisher appears to be partly degraded under nitrogen deprivation (36), Scientific), by incubating iodine-treated cells with 2 µM of the dye. the corresponding acyl chains may return to the ER or to LD membranes for TAG assembly (26). Generation of CrLPAAT2 RNAi Transgenic Strains. Chlamydomonas strains A meaningful role of CrLPAAT2 in TAG biosynthesis in containing an inverted repeat transgene homologous to Cre17.g738350 Chlamydomonas, under nutrient deprivation, is also supported were obtained as described in SI Appendix, SI Materials and Methods, fol- by the enhanced accumulation of nonpolar lipids in the trans- lowing established protocols (5, 48). genic strains overexpressing CrLPAAT2-mCherry (SI Appendix, Figs. S7 and S8). DGTT1, a Chlamydomonas DGAT that cat- Semiquantitative RT-PCR. The expression of CrLPAAT2 was examined by semiquantitative PCR methods, as described previously (5, 19). Primers and alyzes the last step in TAG synthesis, was found to localize PCR conditions are detailed in SI Appendix, SI Materials and Methods. primarily to the ER (and also somewhat to the chloroplast envelope) and to prefer sn-2-C16-DAG over sn-2-C18-DAG CrLPAAT2 Acyltransferase Activity and Competition Assays. Recombinant as the substrate (37). Barring substantial glycerolipid traf- CrLPAAT2 was produced by in vitro transcription/translation in a cell-free ficking from plastid to the ER, CrLPAAT2 could generate wheat-germ system (Biotechrabbit), in accordance with the manufacturer’s directly in ER membranes the prokaryotic precursor preferred protocol. The 14C-palmitoyl-CoA (60 µCi/µmol; PerkinElmer) or 14C-oleoyl- for DGTT1-mediated TAG assembly. However, ER membrane CoA (60 µCi/µmol; PerkinElmer) was incubated with oleoyl-lysophosphatidic glycerolipids, such as diacylglycerol-N,N,N-trimethylhomoserine acid (Avanti Polar Lipids) and recombinant CrLPAAT2 to examine the forma- 14 (DGTS), phosphatidylethanolamine (PE), or phosphatidylinos- tion of C-PA, as described in previous reports (8). Details of the proto- itol (PI), mostly have C18:3 esterified at the sn-2 position col and of the competition assays with unlabeled palmitoyl-CoA (Sigma) or oleoyl-CoA (Sigma) are provided in SI Appendix, SI Materials and Methods. in Chlamydomonas (3). Thus, other acyltransferases, possibly acyl-CoA:lysophospholipid acyltransferase-like enzymes (SI Lipid Analyses. Total lipids from C. reinhardtii or S. cerevisiae were analyzed Appendix, Fig. S2) (38), may synthesize the precursors involved as described in refs. 5, 19, and 49. To identify fatty acids esterified at the in ER membrane glycerolipid assembly. We propose that, in sn-2 position of the glycerol backbone, TAGs were digested with a Rhizopus Chlamydomonas and related microalgae, CrLPAAT2 may syn- oryzae TAG lipase (62305; Sigma), as outlined in SI Appendix, SI Materials thesize prokaryotic glycerolipid precursors mainly for assem- and Methods. bly into TAGs and/or transfer to the plastid whereas other acyltransferase(s) may synthesize “eukaryotic” glycerolipid pre- Complementation of the S. cerevisiae ale1 ∆-slc1 ∆ Mutant Strain. The cursors mainly for assembly into DGTS/PE/PI. This hypothe- Cre17.g738350 coding sequence was cloned into the pYES2.1 TOPO vector sis implies discrimination between intermediates in the TAG (ThermoFisher Scientific), following the manufacturer’s protocol, and trans- and DGTS/PE/PI biosynthesis pathways (possibly through sub- formed into yeast. The procedure, strain crossing, and spore analyses are explained in detail in SI Appendix, SI Materials and Methods; 5-fluoroorotic strate channeling and/or substrate preference among consecu- acid (5-FOA), at 5 mg/mL, was used to cure the plasmid from the yeast strains. tive enzymes within each pathway) and/or subcompartmentation of the two assembly pathways within the ER of microalgae, as ACKNOWLEDGMENTS. We thank Rebecca Cahoon and Rebecca Ros- already suggested in other eukaryotes (39, 40). ton for help with liquid chromatography-mass spectrometry and gas chromatography-flame ionization detection. This work was supported in In summary, our data strongly indicate that CrLPAAT2, a part by a grant from the National Science Foundation (to H.C.). We also chlorophyte-specific acyltransferase, is localized to the ER but, acknowledge the support of the EPSCoR (Established Program to Stimulate unlike canonical eukaryotic LPAATs, prefers C16:0-CoA as Competitive Research) program (EPS-1004094 to W.R.R., E.B.C., and H.C.).

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