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Protein & Cell

Prot Cell    2011, Vol. 2 Issue (6) : 487-496     https://doi.org/10.1007/s13238-011-1061-y      PMID: 21748599
RESEARCH ARTICLE
Interactomic study on interaction between lipid droplets and mitochondria
Jing Pu1,2, Cheol Woong Ha3, Shuyan Zhang1, Jong Pil Jung3, Won-Ki Huh3(), Pingsheng Liu1()
1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2. Graduate University of Chinese Academy of Sciences, Beijing 100101, China; 3. School of Biological Sciences, Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea
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Abstract

An increasing body of evidence shows that the lipid droplet, a neutral lipid storage organelle, plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria. However, the cellular functions and molecular mechanisms of the interaction remain ambiguous. Here we present data from transmission electron microscopy, fluorescence imaging, and reconstitution assays, demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro. Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae, we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes. The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75% of the interactions detected. Interestingly, interactions between 3 pairs of lipid metabolic enzymes were detected. Collectively, these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes, and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.

Keywords peroxisomes      bimolecular fluorescence complementation assay      protein-protein interaction      lipid metabolism      Erg6     
Corresponding Author(s): Huh Won-Ki,Email:wkh@snu.ac.kr (W.-K.Huh); Liu Pingsheng,Email:pliu@ibp.ac.cn (P. Liu)   
Issue Date: 01 June 2011
 Cite this article:   
Jing Pu,Cheol Woong Ha,Shuyan Zhang, et al. Interactomic study on interaction between lipid droplets and mitochondria[J]. Prot Cell, 2011, 2(6): 487-496.
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http://journal.hep.com.cn/pac/EN/10.1007/s13238-011-1061-y
http://journal.hep.com.cn/pac/EN/Y2011/V2/I6/487
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Jing Pu
Cheol Woong Ha
Shuyan Zhang
Jong Pil Jung
Won-Ki Huh
Pingsheng Liu
Fig.1  Interaction between mitochondria and lipid droplets in skeletal muscle cells.
(A) The ultrastructure of rat skeletal muscle tissue was obtained by transmission electron microscopy (TEM). Mito, mitochondrion; LD, lipid droplet. Bar= 200 nm. (B) L6 cells were treated with 200 μmol/L oleate in the growth medium for 12 h and maintained in the growth medium for 3 h after washing oleate off. Mitochondria (upper panel) and lipid droplets (middle panel) were stained with MitoTracker Green FM and LipidTOX Deep Red, respectively. Fluorescence images were obtained using a confocal microscope and 3D reconstructed by software . The contact between lipid droplets and mitochondria were determined by 3D reconstruction movie and indicated by arrows (lower panel). Bar= 5 μm. (C) Proteins were extracted from purified lipid droplets (LD), mitochondria (Mito), cytosol (Cyto) and postnuclear supernatant (PNS) from C57 mouse liver tissues and were resolved by 10% SDS-PAGE. The gel was stained with a Colloidal Blue Staining Kit (upper panel). M, molecular weight marker. Purified lipid droplets and mitochondria were subjected to reconstitution assays. GTPγs (lane 3) or isolated cytosol (lane 4) was added to the reaction system. Total proteins of lipid droplets were isolated from the system and were analyzed by immunoblotting with the indicated antibodies (lower panel).
Fig.1  Interaction between mitochondria and lipid droplets in skeletal muscle cells.
(A) The ultrastructure of rat skeletal muscle tissue was obtained by transmission electron microscopy (TEM). Mito, mitochondrion; LD, lipid droplet. Bar= 200 nm. (B) L6 cells were treated with 200 μmol/L oleate in the growth medium for 12 h and maintained in the growth medium for 3 h after washing oleate off. Mitochondria (upper panel) and lipid droplets (middle panel) were stained with MitoTracker Green FM and LipidTOX Deep Red, respectively. Fluorescence images were obtained using a confocal microscope and 3D reconstructed by software . The contact between lipid droplets and mitochondria were determined by 3D reconstruction movie and indicated by arrows (lower panel). Bar= 5 μm. (C) Proteins were extracted from purified lipid droplets (LD), mitochondria (Mito), cytosol (Cyto) and postnuclear supernatant (PNS) from C57 mouse liver tissues and were resolved by 10% SDS-PAGE. The gel was stained with a Colloidal Blue Staining Kit (upper panel). M, molecular weight marker. Purified lipid droplets and mitochondria were subjected to reconstitution assays. GTPγs (lane 3) or isolated cytosol (lane 4) was added to the reaction system. Total proteins of lipid droplets were isolated from the system and were analyzed by immunoblotting with the indicated antibodies (lower panel).
Fig.2  Proteins involved in the organelle interaction.
(A) The schema of BiFC assay probing lipid droplet and mitochondrion interaction. The interaction between C-terminally VN-tagged mitochondrial protein A and C-terminally VC-tagged lipid droplet protein B brings VC and VN into close proximity resulting in a fluorescent signal. (B) The BiFC assay was performed and signals from fluorescence complementation were monitored by fluorescence microscopy. Diploid yeast cells expressing C-terminally VC-tagged Tgl3 and Faa2 were analyzed for BiFC and used as a negative control (a). The fluorescence images with 3 different signal patterns are shown in b, c and d. The two genes C-terminally tagged with VC or VN are indicated as gene X-VC/gene Y-VN. (cC) The protein-protein interaction network visualized by Cytoscape. The node size was according to node degree. (D) The proteins interacting with Erg6 (upper panel) or Pet10 (lower panel) were catalogued by their function referring to SGD and the percentages of each category are indicated.
Fig.2  Proteins involved in the organelle interaction.
(A) The schema of BiFC assay probing lipid droplet and mitochondrion interaction. The interaction between C-terminally VN-tagged mitochondrial protein A and C-terminally VC-tagged lipid droplet protein B brings VC and VN into close proximity resulting in a fluorescent signal. (B) The BiFC assay was performed and signals from fluorescence complementation were monitored by fluorescence microscopy. Diploid yeast cells expressing C-terminally VC-tagged Tgl3 and Faa2 were analyzed for BiFC and used as a negative control (a). The fluorescence images with 3 different signal patterns are shown in b, c and d. The two genes C-terminally tagged with VC or VN are indicated as gene X-VC/gene Y-VN. (cC) The protein-protein interaction network visualized by Cytoscape. The node size was according to node degree. (D) The proteins interacting with Erg6 (upper panel) or Pet10 (lower panel) were catalogued by their function referring to SGD and the percentages of each category are indicated.
GeneLipid droplet proteins
Erg6Tgl3Faa4Pet10Yor246cOsw5
Pdh1+
Fol1+a+
Nca2+
Yhr003c++
Sam35+
Mcr1++
Pth2++
Om14++++++
Mdv1++
Msp1++
Atp3+++
Pet8+++
Qcr9++
Cox7++
Mdm34+
Tom71+
Mmm1+
Tim54+
Oac1+
Pex30+++
Inp1++
Pex3+
Pex13++
Pex2++
Pex17++
Pex5+
Pex25+
Pex12+
Pex10+
Total24121924
Tab.1  Protein-protein interactions identified by BiFC assay in
GeneLipid droplet proteinsLocalization
Erg6Tgl3Yeh1Faa4Pet10Bsc2Yor246cOsw5SGDGFP
Arc15+a++mitobActin
Jsn1++mitoAmbiguous
Tom22+++mitoAmbiguous
Iml2+++mitocytoc nud
Hfd1+++mitoendoe LDf
Cbr1++mitoER
Ayr1++++++mitoER
Fmp52+++mitoER
Erg9+mitoER
Gsf2+++mitoER
Dpm1++++mitoER
Dic1+++mitoER
Get1++mitoER
Zeo1+mitoER, perig
Dap1+++mitoER, cyto
Isc1+mitoNoh
Ytp1+++mitoNo
Fis1++mito/peroiAmbiguous
Pex31+peroAmbiguous
Pex32+peroAmbiguous
Pex29++peroAmbiguous,
Npy1+perocyto, nu
Pex19+++++perocyto
Dsl1+++peroER
Dyn2+peroER
Djp1++peroER
Total20311241212
Tab.2  Protein-protein interactions identified by BiFC assay in (supplement)
NameMolecular functionBiological process
Erg7Lanosterol synthase activityErgosterol biosynthetic process
Tgl4Sterol esterase activityTriglyceride catabolism
Triglyceride lipase activityTriglyceride mobilization
Yeh1Sterol esterase activitySterol metabolism
Tgl3Triglyceride lipase activityTriglyceride catabolic process
Cellular lipid metabolic process
Coy1UnknownGolgi vesicle transport
Yor246cOxidoreductase activityUnknown
Pet10UnknownAerobic respiration
Rrt8UnknownUnknown
Bsc2UnknownUnknown
Ymr148wUnknownUnknown
Tab.3  Lipid droplet proteins interacting with Erg6
Lipid droplet protein (bait)Mitochondrial or peroxisomal protein (prey)
NameMolecular functionBiological processNameMolecular functionBiological process
Erg6Sterol 24-C-methyltransferaseErgosterol biosynthetic processMcr1Cytochrome b5 reductaseErgosterol biosynthetic process
Tgl3Triglyceride lipase activity Triglyceride catabolic process Cellular lipid metabolic processAyr1Acylglycerone-phosphate reductasePhosphatidic acid biosynthetic
Pex11UnknownFatty acid oxidation
Tab.4  Protein-protein interactions involved in lipid metabolism between lipid droplets and mitochondria or peroxisomes
1 Beller, M., Riedel, D., J?nsch, L., Dieterich, G., Wehland, J., J?ckle, H., and Kühnlein, R.P. (2006). Characterization of the Drosophila lipid droplet subproteome. Mol Cell Proteomics 5, 1082–1094 .
pmid:16543254
2 Binns, D., Januszewski, T., Chen, Y., Hill, J., Markin, V.S., Zhao, Y., Gilpin, C., Chapman, K.D., Anderson, R.G., and Goodman, J.M. (2006). An intimate collaboration between peroxisomes and lipid bodies. J Cell Biol 173, 719–731 .
pmid:16735577
3 Blanchette-Mackie, E.J., and Scow, R.O. (1983). Movement of lipolytic products to mitochondria in brown adipose tissue of young rats: an electron microscope study. J Lipid Res 24, 229–244 .
pmid:6842081
4 Blondel, M., Bach, S., Bamps, S., Dobbelaere, J., Wiget, P., Longaretti, C., Barral, Y., Meijer, L., and Peter, M. (2005). Degradation of Hof1 by SCF(Grr1) is important for actomyosin contraction during cytokinesis in yeast. EMBO J 24, 1440–1452 .
pmid:15775961
5 Brasaemle, D.L., Dolios, G., Shapiro, L., and Wang, R. (2004). Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J Biol Chem 279, 46835–46842 .
pmid:15337753
6 Cermelli, S., Guo, Y., Gross, S.P., and Welte, M.A. (2006). The lipid-droplet proteome reveals that droplets are a protein-storage depot. Curr Biol 16, 1783–1795 .
pmid:16979555
7 Egan, J.J., Greenberg, A.S., Chang, M.K., Wek, S.A., Moos, M.C. Jr, and Londos, C. (1992). Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. Proc Natl Acad Sci U S A 89, 8537–8541 .
pmid:1528859
8 Gaber, R.F., Copple, D.M., Kennedy, B.K., Vidal, M., and Bard, M. (1989). The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol Cell Biol 9, 3447–3456 .
pmid:2677674
9 Goodman, J.M. (2008). The gregarious lipid droplet. J Biol Chem 283, 28005–28009 .
pmid:18611863
10 Guo, Y., Jangi, S., and Welte, M.A. (2005). Organelle-specific control of intracellular transport: distinctly targeted isoforms of the regulator Klar. Mol Biol Cell 16, 1406–1416 .
pmid:15647372
11 Hu, C.D., and Kerppola, T.K. (2003). Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21, 539–545 .
pmid:12692560
12 Huh, W.K., Falvo, J.V., Gerke, L.C., Carroll, A.S., Howson, R.W., Weissman, J.S., and O’Shea, E.K. (2003). Global analysis of protein localization in budding yeast. Nature 425, 686–691 .
pmid:14562095
13 J?gerstr?m, S., Polesie, S., Wickstr?m, Y., Johansson, B.R., Schr?der, H.D., H?jlund, K., and Bostr?m, P. (2009). Lipid droplets interact with mitochondria using SNAP23. Cell Biol Int 33, 934–940 .
pmid:19524684
14 Kalashnikova, M.M., and Fadeeva, E.O. (2006). Ultrastructural study of liver cells from rooks living in ecologically unfavorable areas. Izv Akad Nauk Ser Biol (2), 133–141 .
pmid:16634429
15 Katavic, V., Agrawal, G.K., Hajduch, M., Harris, S.L., and Thelen, J.J. (2006). Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars. Proteomics 6, 4586–4598 .
pmid:16847873
16 Liu, P., Bartz, R., Zehmer, J.K., Ying, Y., and Anderson, R.G. (2008). Rab-regulated membrane traffic between adiposomes and multiple endomembrane systems. Methods Enzymol 439, 327–337 .
pmid:18374175
17 Liu, P., Bartz, R., Zehmer, J.K., Ying, Y.S., Zhu, M., Serrero, G., and Anderson, R.G. (2007). Rab-regulated interaction of early endosomes with lipid droplets. Biochim Biophys Acta 1773, 784–793 .
pmid:17395284
18 Liu, P., Ying, Y., Zhao, Y., Mundy, D.I., Zhu, M., and Anderson, R.G. (2004). Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J Biol Chem 279, 3787–3792 .
pmid:14597625
19 Martin, S., and Parton, R.G. (2006). Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7, 373–378 .
pmid:16550215
20 Murphy, D.J. (2001). The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40, 325–438 .
pmid:11470496
21 Murphy, S., Martin, S., and Parton, R.G. (2009). Lipid droplet-organelle interactions; sharing the fats. Biochim Biophys Acta 1791, 441–447 .
pmid:18708159
22 Novikoff, A.B., Novikoff, P.M., Rosen, O.M., and Rubin, C.S. (1980). Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol 87, 180–196 .
pmid:7191426
23 Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498–2504 .
pmid:14597658
24 Shaw, C.S., Jones, D.A., and Wagenmakers, A.J. (2008). Network distribution of mitochondria and lipid droplets in human muscle fibres. Histochem Cell Biol 129, 65–72 .
pmid:17938948
25 Sung, M.K., and Huh, W.K. (2007). Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae. Yeast 24, 767–775 .
pmid:17534848
26 Tarnopolsky, M.A., Rennie, C.D., Robertshaw, H.A., Fedak-Tarnopolsky, S.N., Devries, M.C., and Hamadeh, M.J. (2007). Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity. Am J Physiol Regul Integr Comp Physiol 292, R1271–R1278 .
pmid:17095651
27 Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R., and Fujimoto, T. (2002). The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition. J Biol Chem 277, 44507–44512 .
pmid:12221100
28 Tedrick, K., Trischuk, T., Lehner, R., and Eitzen, G. (2004). Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement. Mol Biol Cell 15, 4609–4621 .
pmid:15254266
29 Turró, S., Ingelmo-Torres, M., Estanyol, J.M., Tebar, F., Fernández, M.A., Albor, C.V., Gaus, K., Grewal, T., Enrich, C., and Pol, A. (2006). Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets. Traffic 7, 1254–1269 .
pmid:17004324
30 Zehmer, J.K., Huang, Y., Peng, G., Pu, J., Anderson, R.G., and Liu, P. (2009). A role for lipid droplets in inter-membrane lipid traffic. Proteomics 9, 914–921 .
pmid:19160396
31 Zhang, S., Du, Y., Wang, Y., and Liu, P. (2010). Lipid Droplet — A Cellular Organelle for Lipid Metabolism. Acta Biophisica Sinica 26, 97–105 .
32 Zimmermann, R., Strauss, J.G., Haemmerle, G., Schoiswohl, G., Birner-Gruenberger, R., Riederer, M., Lass, A., Neuberger, G., Eisenhaber, F., Hermetter, A., (2004). Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306, 1383–1386 .
pmid:15550674
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