Isolation of Lipid Droplets for Protein and Lipid Analysis.
Confocal microscopy
Lipid droplets
Mass spectrometry
Oil bodies
Plant organelles
Proteomics
Subcellular fractionation
Transient expression
Triacylglycerols
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2021
2021
Historique:
entrez:
28
5
2021
pubmed:
29
5
2021
medline:
23
6
2021
Statut:
ppublish
Résumé
Cytosolic lipid droplets (LDs) are organelles which emulsify a variety of hydrophobic molecules in the aqueous cytoplasm of essentially all plant cells. Most familiar are the LDs from oilseeds or oleaginous fruits that primarily store triacylglycerols and serve a storage function. However, similar hydrophobic particles are found in cells of plant tissues that package terpenoids, sterol esters, wax esters, or other types of nonpolar lipids. The various hydrophobic lipids inside LDs are coated with a phospholipid monolayer, mostly derived from membrane phospholipids during their ontogeny. Various proteins have been identified to be associated with LDs, and these may be cell-type, tissue-type, or even species specific. While major LD proteins like oleosins have been known for decades, more recently a growing list of LD proteins has been identified, primarily by proteomics analyses of isolated LDs and confirmation of their localization by confocal microscopy. LDs, unlike other organelles, have a density less than that of water, and consequently can be isolated and enriched in cellular fractions by flotation centrifugation for composition studies. However, due to its deep coverage, modern proteomics approaches are also prone to identify contaminants, making control experiments necessary. Here, procedures for the isolation of LDs, and analysis of LD components are provided as well as methods to validate the LD localization of proteins.
Identifiants
pubmed: 34047983
doi: 10.1007/978-1-0716-1362-7_16
doi:
Substances chimiques
Lipids
0
Phospholipids
0
Proteins
0
Proteome
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
295-320Subventions
Organisme : Howard Hughes Medical Institute
Pays : United States
Références
Chapman KD, Aziz M, Dyer JM, Mullen RT (2019) Mechanisms of lipid droplet biogenesis. Biochem J 476(13):1929–1942. https://doi.org/10.1042/BCJ20180021
doi: 10.1042/BCJ20180021
pubmed: 31289128
Pyc M, Cai Y, Greer MS, Yurchenko O, Chapman KD, Dyer JM, Mullen RT (2017) Turning over a new leaf in lipid droplet biology. Trends Plant Sci 22(7):596–609. https://doi.org/10.1016/j.tplants.2017.03.012
doi: 10.1016/j.tplants.2017.03.012
pubmed: 28454678
Shimada TL, Hayashi M, Hara-Nishimura I (2018) Membrane dynamics and multiple functions of oil bodies in seeds and leaves. Plant Physiol 176(1):199–207. https://doi.org/10.1104/pp.17.01522
doi: 10.1104/pp.17.01522
pubmed: 29203559
Berthelot K, Lecomte S, Estevez Y, Peruch F (2014) Hevea brasiliensis REF (Hev b 1) and SRPP (Hev b 3): an overview on rubber particle proteins. Biochimie 106:1–9. https://doi.org/10.1016/j.biochi.2014.07.002
doi: 10.1016/j.biochi.2014.07.002
pubmed: 25019490
Zhou X, Chen X, Du Z, Zhang Y, Zhang W, Kong X, Thelen JJ, Chen C, Chen M (2019) Terpenoid esters are the major constituents from leaf lipid droplets of Camellia sinensis. Front Plant Sci 10:179. https://doi.org/10.3389/fpls.2019.00179
doi: 10.3389/fpls.2019.00179
pubmed: 30863415
pmcid: 6399487
Rotsch AH, Kopka J, Feussner I, Ischebeck T (2017) Central metabolite and sterol profiling divides tobacco male gametophyte development and pollen tube growth into eight metabolic phases. Plant J 92(1):129–146. https://doi.org/10.1111/tpj.13633
doi: 10.1111/tpj.13633
pubmed: 28685881
Cai Y, McClinchie E, Price A, Nguyen TN, Gidda SK, Watt SC, Yurchenko O, Park S, Sturtevant D, Mullen RT (2017) Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants. Plant Biotechnol J 15(7):824–836. https://doi.org/10.1111/pbi.12678
doi: 10.1111/pbi.12678
pubmed: 27987528
pmcid: 5466434
Bouvier-Nave P, Berna A, Noiriel A, Compagnon V, Carlsson AS, Banas A, Stymne S, Schaller H (2010) Involvement of the phospholipid sterol acyltransferase1 in plant sterol homeostasis and leaf senescence. Plant Physiol 152(1):107–119. https://doi.org/10.1104/pp.109.145672
doi: 10.1104/pp.109.145672
pubmed: 19923239
pmcid: 2799350
Miwa TK (1971) Jojoba oil wax esters and derived fatty acids and alcohols - Gas chromatographic analyses. J Am Oil Chem Soc 48(6):259–264
doi: 10.1007/BF02638458
Tzen JTC, Cao Y-z, Laurent P, Ratnayake C, Huang AHC (1993) Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiol 101:267–276. https://doi.org/10.1104/pp.101.1.267
doi: 10.1104/pp.101.1.267
pubmed: 12231682
pmcid: 158673
Chapman KD, Trelease RN (1991) Acquisition of membrane lipids by differentiating glyoxysomes – role of lipid bodies. J Cell Biol 115(4):995–1007. https://doi.org/10.1083/jcb.115.4.995
doi: 10.1083/jcb.115.4.995
pubmed: 1955468
Donaldson RP (1977) Membrane lipid metabolism in germinating castor bean endosperm. Plant Physiol 57:510–515. https://doi.org/10.1104/pp.57.4.510
doi: 10.1104/pp.57.4.510
Horn PJ, James CN, Gidda SK, Kilaru A, Dyer JM, Mullen RT, Ohlrogge JB, Chapman KD (2013) Identification of a new class of lipid droplet-associated proteins in plants. Plant Physiol 162(4):1926–1936. https://doi.org/10.1104/pp.113.222455
doi: 10.1104/pp.113.222455
pubmed: 23821652
pmcid: 3729771
Moellering ER, Benning C (2010) RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell 9(1):97–106. https://doi.org/10.1128/ec.00203-09
doi: 10.1128/ec.00203-09
pubmed: 19915074
Lin IP, Jiang P-L, Chen C-S, Tzen JTC (2012) A unique caleosin serving as the major integral protein in oil bodies isolated from Chlorella sp. cells cultured with limited nitrogen. Plant Physiol Biochem 61(0):80–87. https://doi.org/10.1016/j.plaphy.2012.09.008
doi: 10.1016/j.plaphy.2012.09.008
pubmed: 23085585
Siloto RMP, Findlay K, Lopez-Villalobos A, Yeung EC, Nykiforuk CL, Moloney MM (2006) The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis. Plant Cell 18(8):1961–1974. https://doi.org/10.1105/tpc.106.041269
doi: 10.1105/tpc.106.041269
pubmed: 16877495
pmcid: 1533971
Taurino M, Costantini S, De Domenico S, Stefanelli F, Ruano G, Delgadillo MO, Sanchez-Serrano JJ, Sanmartín M, Santino A, Rojo E (2017) SEIPIN proteins mediate lipid droplet biogenesis to promote pollen transmission and reduce seed dormancy. Plant Physiol 176(2):1531–1546. https://doi.org/10.1104/pp.17.01430
doi: 10.1104/pp.17.01430
pubmed: 29203558
pmcid: 5813562
Horn PJ, Ledbetter NR, James CN, Hoffman WD, Case CR, Verbeck GF, Chapman KD (2011) Visualization of lipid droplet composition by direct organelle mass spectrometry. J Biol Chem 286(5):3298–3306. https://doi.org/10.1074/jbc.M110.186353
doi: 10.1074/jbc.M110.186353
pubmed: 21118810
Murphy DJ, Cummins I (1989) Seed oil-bodies: isolation, composition and role of oil-body apolipoproteins. Phytochemistry 28(8):2063–2069. https://doi.org/10.1016/S0031-9422(00)97921-4
doi: 10.1016/S0031-9422(00)97921-4
Ding Y, Zhang S, Yang L, Na H, Zhang P, Zhang H, Wang Y, Chen Y, Yu J, Huo C, Xu S, Garaiova M, Cong Y, Liu P (2013) Isolating lipid droplets from multiple species. Nat Protoc 8(1):43–51. https://doi.org/10.1038/nprot.2012.142
doi: 10.1038/nprot.2012.142
pubmed: 23222457
Mannik J, Meyers A, Dalhaimer P (2014) Isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas. J Vis Exp 86:50981. https://doi.org/10.3791/50981
doi: 10.3791/50981
Brasaemle DL, Wolins NE (2016) Isolation of lipid droplets from cells by density gradient centrifugation. Curr Protoc Cell Biol 72:3.15.11–13.15.13. https://doi.org/10.1002/cpcb.10
doi: 10.1002/cpcb.10
Kretzschmar FK, Doner N, Krawczyk HE, Scholz P, Schmitt K, Valerius O, Braus G, Mullen RT, Ischebeck T (2020) Identification of low-abundance lipid droplet proteins in seeds and seedlings. Plant Physiol 182(3):1236–1245. https://doi.org/10.1104/pp.19.01255
doi: 10.1104/pp.19.01255
Kretzschmar FK, Mengel LF, Müller A, Schmitt K, Blersch KF, Valerius O, Braus G, Ischebeck T (2018) PUX10 is a lipid droplet-localized scaffold protein that interacts with CDC48 and is involved in the degradation of lipid droplet proteins. Plant Cell 30:2137–2160. https://doi.org/10.1105/tpc.18.00276
doi: 10.1105/tpc.18.00276
pubmed: 30087207
pmcid: 6181012
Brocard L, Immel F, Coulon D, Esnay N, Tuphile K, Pascal S, Claverol S, Fouillen L, Bessoule JJ, Brehelin C (2017) Proteomic analysis of lipid droplets from Arabidopsis aging leaves brings new insight into their biogenesis and functions. Front Plant Sci 8:894. https://doi.org/10.3389/fpls.2017.00894
doi: 10.3389/fpls.2017.00894
pubmed: 28611809
pmcid: 5447075
Lupette J, Jaussaud A, Seddiki K, Morabito C, Brugière S, Schaller H, Kuntz M, Putaux J-L, Jouneau P-H, Rébeillé F, Falconet D, Coute Y, Jouhet J, Tardif M, Salvaing J, Maréchal E (2019) The architecture of lipid droplets in the diatom Phaeodactylum tricornutum. Algal Res 38:101415. https://doi.org/10.1016/j.algal.2019.101415
doi: 10.1016/j.algal.2019.101415
Huang AHC (1992) Oil bodies and oleosins in seeds. Annu Rev Plant Physiol Plant Mol Biol 43:177–200. https://doi.org/10.1146/annurev.pp.43.060192.001141
doi: 10.1146/annurev.pp.43.060192.001141
Vieler A, Brubaker SB, Vick B, Benning C (2012) A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins. Plant Physiol 158(4):1562–1569. https://doi.org/10.1104/pp.111.193029
doi: 10.1104/pp.111.193029
pubmed: 22307965
pmcid: 3320170
Sadre R, Kuo P, Chen J, Yang Y, Banerjee A, Benning C, Hamberger B (2019) Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves. Nat Commun 10(1):853. https://doi.org/10.1038/s41467-019-08515-4
doi: 10.1038/s41467-019-08515-4
pubmed: 30787273
pmcid: 6382807
Kim EY, Park KY, Seo YS, Kim WT (2016) Arabidopsis small rubber particle protein homolog SRPs play dual roles as positive factors for tissue growth and development and in drought stress responses. Plant Physiol 170(4):2494–2510. https://doi.org/10.1104/pp.16.00165
doi: 10.1104/pp.16.00165
pubmed: 26903535
pmcid: 4825120
Gidda SK, Park S, Pyc M, Yurchenko O, Cai Y, Wu P, Andrews DW, Chapman KD, Dyer JM, Mullen RT (2016) Lipid droplet-associated proteins (LDAPs) are required for the dynamic regulation of neutral lipid compartmentation in plant cells. Plant Physiol 170(4):2052–2071. https://doi.org/10.1104/pp.15.01977
doi: 10.1104/pp.15.01977
pubmed: 26896396
pmcid: 4825156
Pyc M, Cai Y, Gidda SK, Yurchenko O, Park S, Kretzschmar FK, Ischebeck T, Valerius O, Braus GH, Chapman KD, Dyer JM, Mullen RT (2017) Arabidopsis lipid drop-associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds. Plant J 92(6):1182–1201. https://doi.org/10.1111/tpj.13754
doi: 10.1111/tpj.13754
pubmed: 29083105
Coulon D, Brocard L, Tuphile K, Brehelin C (2020) Arabidopsis LDIP protein locates at a confined area within the lipid droplet surface and favors lipid droplet formation. Biochimie 169:29–40. https://doi.org/10.1016/j.biochi.2019.09.018
doi: 10.1016/j.biochi.2019.09.018
pubmed: 31568826
Feussner I, Balkenhohl TJ, Porzel A, Kühn H, Wasternack C (1997) Structural elucidation of oxygenated storage lipids in cucumber cotyledons – implication of lipid body lipoxygenase in lipid mobilization during germination. J Biol Chem 272(34):21635–21641. https://doi.org/10.1074/jbc.272.34.21635
doi: 10.1074/jbc.272.34.21635
pubmed: 9261186
Shimada TL, Takano Y, Shimada T, Fujiwara M, Fukao Y, Mori M, Okazaki Y, Saito K, Sasaki R, Aoki K, Hara-Nishimura I (2014) Leaf oil body functions as a subcellular factory for the production of a phytoalexin in Arabidopsis. Plant Physiol 164(1):105–118. https://doi.org/10.1104/pp.113.230185
doi: 10.1104/pp.113.230185
pubmed: 24214535
Müller AO, Ischebeck T (2018) Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1. New Phytol 217(3):1062–1076. https://doi.org/10.1111/nph.14902
doi: 10.1111/nph.14902
pubmed: 29178188
Eastmond PJ (2004) Cloning and characterization of the acid lipase from castor beans. J Biol Chem 279(44):45540–45545. https://doi.org/10.1074/jbc.M408686200
doi: 10.1074/jbc.M408686200
pubmed: 15322116
Garbowicz K, Liu Z, Alseekh S, Tieman D, Taylor M, Kuhalskaya A, Ofner I, Zamir D, Klee HJ, Fernie AR, Brotman Y (2018) Quantitative trait loci analysis identifies a prominent gene involved in the production of fatty-acid-derived flavor volatiles in tomato. Mol Plant 11:1147–1165. https://doi.org/10.1016/j.molp.2018.06.003
doi: 10.1016/j.molp.2018.06.003
pubmed: 29960108
Deruyffelaere C, Purkrtova Z, Bouchez I, Collet B, Cacas JL, Chardot T, Gallois JL, D'Andrea S (2018) PUX10 associates with CDC48A and regulates the dislocation of ubiquitinated oleosins from seed lipid droplets. Plant Cell 30(9):2116–2136. https://doi.org/10.1105/tpc.18.00275
doi: 10.1105/tpc.18.00275
pubmed: 30087208
pmcid: 6181022
Gallagher SR (2006) One-dimensional SDS gel electrophoresis of proteins. Curr Prot Mol Biol 75(1):10.12.11–10.12A.37. https://doi.org/10.1002/0471142727.mb1002as75
doi: 10.1002/0471142727.mb1002as75
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860. https://doi.org/10.1038/nprot.2006.468
doi: 10.1038/nprot.2006.468
pubmed: 17406544
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896. https://doi.org/10.1038/nprot.2007.261
doi: 10.1038/nprot.2007.261
pubmed: 17703201
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367. https://doi.org/10.1038/nbt.1511
doi: 10.1038/nbt.1511
pubmed: 19029910
Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513–2526. https://doi.org/10.1074/mcp.M113.031591
doi: 10.1074/mcp.M113.031591
pubmed: 24942700
pmcid: 4159666
Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740. https://doi.org/10.1038/nmeth.3901
doi: 10.1038/nmeth.3901
pubmed: 27348712
Krishnakumar V, Hanlon MR, Contrino S, Ferlanti ES, Karamycheva S, Kim M, Rosen BD, Cheng C-Y, Moreira W, Mock SA, Stubbs J, Sullivan JM, Krampis K, Miller JR, Micklem G, Vaughn M, Town CD (2014) Araport: the Arabidopsis information portal. Nucleic Acids Res 43(D1):D1003–D1009. https://doi.org/10.1093/nar/gku1200
doi: 10.1093/nar/gku1200
pubmed: 25414324
pmcid: 4383980
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133(2):462–469. https://doi.org/10.1104/pp.103.027979
doi: 10.1104/pp.103.027979
pubmed: 14555774
pmcid: 523872
Petrie JR, Shrestha P, Liu Q, Mansour MP, Wood CC, Zhou XR, Nichols PD, Green AG, Singh SP (2010) Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production. Plant Methods 6:8. https://doi.org/10.1186/1746-4811-6-8
doi: 10.1186/1746-4811-6-8
pubmed: 20222981
pmcid: 2845569
Mähs A, Steinhorst L, Han JP, Shen LK, Wang Y, Kudla J (2013) The calcineurin B-like Ca2+ sensors CBL1 and CBL9 function in pollen germination and pollen tube growth in Arabidopsis. Mol Plant 6(4):1149–1162. https://doi.org/10.1093/mp/sst095
doi: 10.1093/mp/sst095
pubmed: 23741064
Steinhorst L, Mahs A, Ischebeck T, Zhang C, Zhang X, Arendt S, Schultke S, Heilmann I, Kudla J (2015) Vacuolar CBL-CIPK12 Ca
doi: 10.1016/j.cub.2015.03.053
pubmed: 25936548
Müller AO, Blersch KF, Gippert AL, Ischebeck T (2017) Tobacco pollen tubes – a fast and easy tool to study lipid droplet association of plant proteins. Plant J 89(5):1055–1064. https://doi.org/10.1111/tpj.13441
doi: 10.1111/tpj.13441
pubmed: 27943529
Read S, Clarke A, Bacic A (1993) Stimulation of growth of cultured Nicotiana tabacum W 38 pollen tubes by poly (ethylene glycol) and Cu (II) salts. Protoplasma 177(1–2):1–14
doi: 10.1007/BF01403393
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. https://doi.org/10.1007/BF01403393
doi: 10.1007/BF01403393
pubmed: 13671378
pmcid: 13671378
Horn PJ, Chapman KD (2012) Lipidomics in tissues, cells and subcellular compartments. Plant J 70(1):69–80. https://doi.org/10.1111/j.1365-313X.2011.04868.x
doi: 10.1111/j.1365-313X.2011.04868.x
pubmed: 22117762
Wang Z, Benning C (2011) Arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (TLC) coupled with gas-liquid chromatography (GLC). J Vis Exp 49:e2518. https://doi.org/10.3791/2518
doi: 10.3791/2518
Christie WW, Han X (2010) Lipid analysis – isolation, separation, identification and lipidomic analysis, 4th edn. Oily Press, Bridgwater
Mahmood T, Yang P-C (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4(9):429–434. https://doi.org/10.4103/1947-2714.100998
doi: 10.4103/1947-2714.100998
pubmed: 23050259
pmcid: 3456489
Schmitt K, Smolinski N, Neumann P, Schmaul S, Hofer-Pretz V, Braus GH, Valerius O (2017) Asc1p/RACK1 connects ribosomes to eukaryotic phosphosignaling. Mol Cell Biol 37(3):e00279–e00216. https://doi.org/10.1128/MCB.00279-16
doi: 10.1128/MCB.00279-16
pubmed: 27821475
pmcid: 5247610
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Prot 1(4):2019–2025. https://doi.org/10.1038/nprot.2006.286
doi: 10.1038/nprot.2006.286
Cai Y, Goodman JM, Pyc M, Mullen RT, Dyer JM, Chapman KD (2015) Arabidopsis SEIPIN proteins modulate triacylglycerol accumulation and influence lipid droplet proliferation. Plant Cell 27(9):2616–2636. https://doi.org/10.1105/tpc.15.00588
doi: 10.1105/tpc.15.00588
pubmed: 26362606
pmcid: 4815042
Dracopoli NC, Haines JL, Korf BR, Moir T, Morton CC, Seidman CE et al (eds) (1994) Current protocols in human genetics. Wiley, Hoboken, NJ. ISBN:978-0-471-03420
Kost B, Lemichez E, Spielhofer P, Hong Y, Tolias K, Carpenter C, Chua N-H (1999) Rac homologues and compartmentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol 145(2):317–330. https://doi.org/10.1083/jcb.145.2.317
doi: 10.1083/jcb.145.2.317
pubmed: 10209027
pmcid: 2133117
Phelps MS, Verbeck GF (2020) Analysis of lipids in single cells and organelles using nanomanipulation-coupled mass spectrometry. In: Shrestha B (ed) Single cell metabolism: methods and protocols. Springer New York, New York, NY, pp 19–30. https://doi.org/10.1007/978-1-4939-9831-9_3
doi: 10.1007/978-1-4939-9831-9_3