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Subcellular localization and identification of acyl-CoA: lysophosphatidylethanolamine acyltransferase (LPEAT) in the arachidonic acid-rich green microalga, Myrmecia incisa Reisigl

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Abstract

Lysophosphatidylethanolamine acyltransferase (LPEAT) plays an important role in acyl remodeling of phospholipid via the Lands’ cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In the present study, MiLPEAT was cloned from an arachidonic acid (ArA)-rich green microalga Myrmecia incisa. Its full-length cDNA was 1,303 bp containing a 981-bp open reading frame that encoded a 326-amino acid protein. Comparing the cDNA to its corresponding cloned DNA sequence showed that MiLPEAT possessed 6 introns. Bioinformatics analysis of LPEAT indicated that a phosphate acyltransferase domain, PlsC, consisting of 4 typical motifs, NH(x)4D, GCxYVxR, FPEGT, and PVxPVx, was present at the C-terminus of MiLPEAT while two (at least one) transmembrane domains at the N-terminus. The cDNA corresponding to C-terminal 224-residues was thereby subcloned into the vectors pET-28a and pMAL-c2X independently for production of recombinant MiLPEAT (rMiLPEAT). The purified soluble rMiLPEAT fused with maltose-binding protein was used for enzyme assay, and thin-layer chromatography profiles of the catalytic products demonstrated that rMiLPEAT could acylate lysophosphatidylethanolamine to phosphatidylethanolamine, thus functionally identifying MiLPEAT. Anti-MiLPEAT polyclonal antibody was generated against the purified rMiLPEAT fused with 6×His tag. Immuno-electron microscopic results with this polyclonal antibody illustrated that MiLPEAT was localized on M. incisa plasma membrane, and this was further supported by immunocytochemical observations. A di-lysine motif present at the C-terminus implying that MiLPEAT was an endoplasmic reticulum resident, how MiLPEAT was transported to the microalgal plasma membrane was therefore discussed. This study will lay a foundation to understand that how M. incisa uses ArA to synthesize TAG.

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Data availability

The datasets generated during and/or analyzed during the current study are available in the GenBank repository [https://www.ncbi.nlm.nih.gov/nuccore/mg558460].

References

  • Akopian D, Shen K, Zhang X, Shan S (2013) Signal recognition particle: an essential protein-targeting machine. Annu Rev Biochem 82:693–721

  • Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochim Biophys Acta 1861:1214–1225

    Article  CAS  PubMed  Google Scholar 

  • Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem 31:101–109

    Article  CAS  PubMed  Google Scholar 

  • Bernal M, Testillano PS, Alfonso M, del Carmen RM, Picorel R, Yruela I (2007) Identification and subcellular localization of the soybean copper P1B-ATPase GmHMA8 transporter. J Struct Biol 158:46–58

    Article  CAS  PubMed  Google Scholar 

  • Béthune J, Wieland F, Moelleken J (2006)COPI-mediated transport. J Membr Biol 211:65–79

    Article  PubMed  Google Scholar 

  • Bi Y-H, Qiao Y-M, Wang Z, Zhou Z-G(2021) Identification and characterization of a periplasmic α-carbonic anhydrase (CA) in the gametophytes of Saccharina japonica (Phaeophyceae). J Phycol 57:295–310

    Article  CAS  PubMed  Google Scholar 

  • Bigogno C, Khozin-Goldberg I, Boussiba S, Vonshak A, Cohen Z (2002) Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid. Phytochemistry 60:497–503

    Article  CAS  PubMed  Google Scholar 

  • Bowie JU (1997) Helix packing in membrane proteins. J Mol Biol 272:780–789

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chen C-X, Sun Z, Cao H-S, Fang F-L, Ouyang L-L, Zhou Z-G(2015) Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl. Algal Res 12:280–288

    Article  Google Scholar 

  • Dyer JM, Stymne S, Green AG, Carlsson AS (2008)High-value oils from plants. Plant J 54:640–655

    Article  CAS  PubMed  Google Scholar 

  • Gemmer M, Förster F (2020) A clearer picture of the ER translocon complex. J Cell Sci 133:jcs231340

    Article  CAS  PubMed  Google Scholar 

  • Goring DR, Di Sansebastiano GP (2018) Protein and membrane trafficking routes in plants: conventional or unconventional? J Exp Bot 69:1–5

    Article  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  CAS  PubMed  Google Scholar 

  • Hishikawa D, Hashidate T, Shimizu T, Shindou H (2014) Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells. J Lipid Res 55:799–807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu Y-J, Zhou Z-G(2001) Extraction of RAPD-friendly DNA from Laminaria japonica(Phaeophyta) after enzymatic dissociation of the frozen sporophyte tissue. J Appl Phycol 13:415–422

    Article  CAS  Google Scholar 

  • Jackowski S, Jackson PD, Rock CO (1994) Sequence and function of the aas gene in Escherichia coli. J Biol Chem 269:2921–2928

    Article  CAS  PubMed  Google Scholar 

  • Jasieniecka-Gazarkiewicz K, Demski K, Gidda SK, Klińska S, Niedojadło J, Lager I, Carlsson AS, Minina EA, Mullen RT, Bozhkov PV, Stymne S, Banaś A (2021) Subcellular localization of acyl-CoA: lysophosphatidylethanolamine acyltransferases (LPEATs) and the effects of knocking-out and overexpression of their genes on autophagy markers level and life span of A. thaliana. Int J Mol Sci 22:3006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jasieniecka-Gazarkiewicz K, Demski K, Lager I, Stymne S, Banaś A (2016) Possible role of different yeast and plant lysophospholipid: acyl-CoA acyltransferases (LPLAT) in acyl remodeling of phospholipids. Lipids 51:15–32

    Article  CAS  PubMed  Google Scholar 

  • Jasieniecka-Gazarkiewicz K, Lager I, Carlsson AS, Gutbrod K, Peisker H, Dörmann P, Stymne S, Banaś A (2017) Acyl-CoA: lysophosphatidylethanolamine acyltransferase activity regulates growth of Arabidopsis. Plant Physiol 174:986–998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Johnson AE, van Waes MA (1999) The translocon: A dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 15:799–842

    Article  PubMed  Google Scholar 

  • Jürgens G (2004) Membrane trafficking in plants. Annu Rev Cell Dev Biol 20:481–504

    Article  PubMed  Google Scholar 

  • Klińska S, Demski K, Jasieniecka-Gazarkiewicz K, Banaś A (2021) LPEATs tailor plant phospholipid composition through adjusting substrate preferences to temperature. Int J Mol Sci 22:8137

    Article  PubMed  PubMed Central  Google Scholar 

  • Klińska S, Jasieniecka-Gazarkiewicz K, Demski K, Banaś A (2020) Editing of phosphatidic acid and phosphatidylethanolamine by acyl-CoA: lysophospholipid acyltransferases in develo** Camelina sativa seeds. Planta 252:4

    Article  PubMed  PubMed Central  Google Scholar 

  • Krenacs L, Krenacs T, Stelkovics E, Raffeld M (2010)Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol 588:103–119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kussmann M, Roepstorff P (2000) Sample preparation techniques for peptides and proteins analyzed by MALDI-MS. Methods Mol Biol 146:405–424

    CAS  PubMed  Google Scholar 

  • Laemmli UR (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lands WEM (1958) Metabolism of glycerolipides: a comparison of lecithin and triglyceride synthesis. J Biol Chem 231:883–888

    Article  CAS  PubMed  Google Scholar 

  • Larsson KE, Kjellberg JM, Tjellström H, Sandelius AS (2007) LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum. BMC Plant Biol 7:64

    Article  PubMed  PubMed Central  Google Scholar 

  • Letourneur F, Hennecke S, Démollière C, Cosson P (1995) Steric masking of a dilysine endoplasmic reticulum retention motif during assembly of the human high affinity receptor for immunoglobulin E. J Cell Biol 129:971–978

    Article  CAS  PubMed  Google Scholar 

  • Lewin TM, Wang P, Coleman RA (1999) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38:5764–5771

    Article  CAS  PubMed  Google Scholar 

  • Liu W, Sun Z, Chen C-X, Liu J-G, Zhou Z-G(2021) Novel insights into type-2 diacylglycerol acyltransferases in microalga Myrmecia incisa. J Appl Phycol 33:25–35

    Article  CAS  Google Scholar 

  • Liu X-Y, Ouyang L-L, Zhou Z-G(2016) Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress. Sci Rep 6:26610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Napier JA (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol 58:295–319

    Article  CAS  PubMed  Google Scholar 

  • Nesvizhskii AI (2007) Protein identification by tandem mass spectrometry and sequence database searching. Methods Mol Biol 367:87–119

    CAS  PubMed  Google Scholar 

  • Nilsson T, Jackson M, Peterson PA (1989) Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 58:707–718

    Article  CAS  PubMed  Google Scholar 

  • Ogunniyi DS (2006) Castor oil: a vital industrial raw material. Bioresour Technol 97:1086–1091

    Article  CAS  PubMed  Google Scholar 

  • Olsen RE, Henderson RJ (1989) The rapid analysis of neutral and polar marine lipids using double-development HPTLC and scanning densitometry. J Exp Mar Biol Ecol 129:189–197

    Article  CAS  Google Scholar 

  • Osborne AR, Rapoport TA, van den Berg B (2005) Protein translocation by the Sec61/SecY channel. Annu Rev Cell Dev Biol 21:529–550

    Article  CAS  PubMed  Google Scholar 

  • Ouyang L-L, Chen S-H, Li Y, Zhou Z-G(2013a) Transcriptome analysis reveals unique C4-like photosynthesis and oil body formation in an arachidonic acid-rich microalga Myrmecia incisa Reisigl H4301. BMC Genomics 14:396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ouyang L-L, Du D-H, Yu S-Y, Li C-Y, Zhang C-W, Gao H-J, Zhou Z-G(2012) Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta). Chin Sci Bull 57:3342–3352

    Article  CAS  Google Scholar 

  • Ouyang L-L, Li H, Liu F, Tong M, Yu S-Y, Zhou Z-G(2013b) Accumulation of arachidonic acid in a green microalga, Myrmecia incisa, enhanced by nitrogen starvation and its molecular regulation mechanisms. In: Dumancas GG, Murdianti BS, Lucas EA (eds) Arachidonic Acid: Dietary Sources and General Functions. Nova Science Publishers, New York, pp 1–20

    Google Scholar 

  • Ouyang L-L, Li H, Yan X-J, Xu J-L, Zhou Z-G(2016a)Site-directed mutagenesis from Arg195 to His of a microalgal putatively chloroplastidial glycerol-3-phosphate acyltransferase causes an increase of phospholipid level in yeast. Front Plant Sci 7:286

    Article  PubMed  PubMed Central  Google Scholar 

  • Ouyang L-L, Li X-L, Zhou Z-G(2016b) Alternations of photosynthetic membrane lipids and triacylglycerol and their fatty acids in Myrmecia incisa grown in a shift from nitrogen starvation to replenishment. Mar Fish 38:653–662 (in Chinese with English abstract)

    Google Scholar 

  • Rancour DM, Backues SK, Bednarek SY (2010) Protein antigen expression in Escherichia coli for antibody production. Methods Mol Biol 657:3–20

    Article  CAS  PubMed  Google Scholar 

  • Rapoport TA, Goder V, Heinrich SU, Matlack KES (2004)Membrane-protein integration and the role of the translocation channel. Trends Cell Biol 14:568–575

    Article  CAS  PubMed  Google Scholar 

  • Reisigl H (1964) Zur Systematik und Ökologie alpiner Bodenalgen. Österr Bot Z 111:402–499

    Article  Google Scholar 

  • Riekhof WR, Wu J, Jones JL, Voelker DR (2007) Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282:28344–28352

    Article  CAS  PubMed  Google Scholar 

  • Shindou H, Hishikawa D, Harayama T, Eto M, Shimizu T (2013) Generation of membrane diversity by lysophospholipid acyltransferases. J Biochem 154:21–28

    Article  CAS  PubMed  Google Scholar 

  • Shockey J, Regmi A, Cotton K, Adhikari N, Browse J, Bates PD (2016) Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in triacylglycerol biosynthesis. Plant Physiol 170:163–179

    Article  CAS  PubMed  Google Scholar 

  • Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99:303–310

    Article  CAS  PubMed  Google Scholar 

  • Sperling P, Linscheid M, Stöcker S, Mühlbach HP, Heinz E (1993)In vivo desaturation of cis-Δ-9-monounsaturated to cis-Δ9,12-diunsaturated alkenylether glycerolipides. J Biol Chem 268:26935–26940

    Article  CAS  PubMed  Google Scholar 

  • Spiess M, Junne T, Janoschke M (2019) Membrane protein integration and topogenesis at the ER. Protein J 38:306–316

    Article  CAS  PubMed  Google Scholar 

  • Stanier RY, Kunisawa R, Mandel M, Cohen-Bazir G (1971) Purification and properties of unicellular blue-green algae (Order Chlorococcales). Bacteriol Rev 35:171–205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ståhl U, Stålberg K, Stymne S, Ronne H (2008) A family of eukaryotic lysophospholipid acyltransferases with broad specificity. FEBS Lett 582:305–309

    Article  PubMed  Google Scholar 

  • Stålberg K, Ståhl U, Stymne S, Ohlrogge J (2009) Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine. BMC Plant Biol 9:60

    Article  PubMed  PubMed Central  Google Scholar 

  • Stymne S, Appelqvist L-Å (1978) Biosynthesis of linoleate from oleoyl-CoA via oleoyl-phosphatidylcholine in microsomes of develo** safflower seeds. Eur J Biochem 90:223–229

    Article  CAS  PubMed  Google Scholar 

  • Sun L-P, Ouyang L-L, Bao H, Liu J-G, Sun Z, Zhou Z-G(2021) Comparison between two isoforms of glycerol-3-phosphate acyltransferase in microalga Myrmecia incisa: subcellular localization and role in triacylglycerol synthesis. Algal Res 54:102172

    Article  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walker IH, Hsieh P-c, Riggs PD (2010) Mutations in maltose-binding protein that alter affinity and solubility properties. Appl Microbiol Biotechnol 88:187–197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang X, Chung KP, Lin W, Jiang L (2018) Protein secretion in plants: conventional and unconventional pathways and new techniques. J Exp Bot 69:21–37

    Article  Google Scholar 

  • Weier D, Müller C, Gaspers C, Frentzen M (2005) Characterisation of acyltransferases from Synechocystis sp. PCC6803. Biochem Biophys Res Commun 334:1127–1134

    Article  CAS  PubMed  Google Scholar 

  • Yamashita A, Hayashi Y, Nemoto-Sasaki Y, Ito M, Oka S, Tanikawa T, Waku K, Sugiura T (2014) Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms. Prog Lipid Res 53:18–81

    Article  CAS  PubMed  Google Scholar 

  • Yamashita A, Nakanishi H, Suzuki H, Kamata R, Tanaka K, Waku K, Sugiura T (2007) Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1. Biochim Biophys Acta 1771:1202–1215

    Article  CAS  PubMed  Google Scholar 

  • Ye R-X, Yu Z, Shi W-W, Gao H-J, Bi Y-H, Zhou Z-G(2014) Characterization of α-type carbonic anhydrase (CA) gene and subcellular localization of α-CA in the gametophytes of Saccharina japonica. J Appl Phycol 26:881–890

    Article  CAS  Google Scholar 

  • Yu S-Y, Li H, Tong M, Ouyang L-L, Zhou Z-G(2012) Identification of a Δ6 fatty acid elongase gene for arachidonic acid biosynthesis localized to the endoplasmic reticulum in the green microalga Myrmecia incisa Reisigl. Gene 493:219–227

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Jia B, Tan X, Thammina CS, Long H, Liu M, Wen S, Song X, Cao H (2014) Fatty acid profile and unigene-derived simple sequence repeat markers in tung tree (Vernicia fordii). PLoS One 9:e105298

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (grant No. 31772821 to Z.-G. Z.) and the State Double First-class Discipline Project of Aquaculture (to Z.-G. Z.).

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  1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Conferred by the Ministry of Education of China, Shanghai Ocean University, Shanghai, 201306, China

    Yi Liu, Hong Bao, Mei-Li Zhu & Cheng-** Hu

  2. International Research Center for Marine Biosciences Conferred by the Ministry of Science and Technology of China, Shanghai Ocean University, Shanghai, 201306, China

    Zhi-Gang Zhou

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  1. Yi Liu
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  2. Hong Bao
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  3. Mei-Li Zhu
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Liu, Y., Bao, H., Zhu, ML. et al. Subcellular localization and identification of acyl-CoA: lysophosphatidylethanolamine acyltransferase (LPEAT) in the arachidonic acid-rich green microalga, Myrmecia incisa Reisigl. J Appl Phycol 34, 837–855 (2022). https://doi.org/10.1007/s10811-021-02681-z

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