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The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein–protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.
Abul-Husn NS, Cheng X, Li AH, Xin Y, Schurmann C, Stevis P, Liu Y, Kozlitina J, Stender S, Wood GC, Stepanchick AN, Still MD, McCarthy S, O'Dushlaine C, Packer JS, Balasubramanian S, Gosalia N, Esopi D, Kim SY, Mukherjee S, Lopez AE, Fuller ED, Penn J, Chu X, Luo JZ, Mirshahi UL, Carey DJ, Still CD, Feldman MD, Small A, Damrauer SM, Rader DJ, Zambrowicz B, Olson W, Murphy AJ, Borecki IB, Shuldiner AR, Reid JG, Overton JD, Yancopoulos GD, Hobbs HH, Cohen JC, Gottesman O, Teslovich TM, Baras A, Mirshahi T, Gromada J, Dewey FE (2018) A protein-truncating HSD17B13 variant and protection from chronic liver disease. N Engl J Med 378(12): 1096−1106
Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein SD, Daum G (1999) Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J Bacteriol 181(20): 6441−6448
Bartz R, Zehmer JK, Zhu M, Chen Y, Serrero G, Zhao Y, Liu P (2007) Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. J Proteome Res 6(8): 3256−3265
Beller M, Riedel D, Jansch L, Dieterich G, Wehland J, Jackle H, Kuhnlein RP (2006) Characterization of the Drosophila lipid droplet subproteome. Mol Cell Proteomics 5(6): 1082−1094
Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, Londos C (1997) Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38(11): 2249−2263
Bruckner A, Polge C, Lentze N, Auerbach D, Schlattner U (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10(6): 2763−2788
Deng Y, Zhou C, Mirza AH, Bamigbade AT, Zhang S, Xu S, Liu P (2021) Rab18 binds PLIN2 and ACSL3 to mediate lipid droplet dynamics. Biochim Biophys Acta Mol Cell Biol Lipids 1866(7): 158923. https://doi.org/10.1016/j.bbalip.2021.158923
Ding Y, Yang L, Zhang S, Wang Y, Du Y, Pu J, Peng G, Chen Y, Zhang H, Yu J, Hang H, Wu P, Yang F, Yang H, Steinbuchel A, Liu P (2012) Identification of the major functional proteins of prokaryotic lipid droplets. J Lipid Res 53(3): 399−411
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
Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC, Jr., Londos C (1992) Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. Proc Natl Acad Sci USA 89(18): 8537−8541
Einarson MB, Pugacheva EN, Orlinick JR (2007) GST pull-down. CSH Protoc 2007: pdb.prot4757. https://doi.org/10.1101/pdb.prot4757
Evans IM, Paliashvili K (2022) Co-immunoprecipitation assays. Methods Mol Biol 2475: 125−132
Farese RV, Jr., Walther TC (2009) Lipid droplets finally get a little R-E-S-P-E-C-T. Cell 139(5): 855−860
Fujimoto T, Ohsaki Y, Cheng J, Suzuki M, Shinohara Y (2008) Lipid droplets: a classic organelle with new outfits. Histochem Cell Biol 130(2): 263−279
Fujimoto Y, Itabe H, Kinoshita T, Homma KJ, Onoduka J, Mori M, Yamaguchi S, Makita M, Higashi Y, Yamashita A, Takano T (2007) Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7. J Lipid Res 48(6): 1280−1292
Fujimoto Y, Itabe H, Sakai J, Makita M, Noda J, Mori M, Higashi Y, Kojima S, Takano T (2004) Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim Biophys Acta 1644(1): 47−59
Gnanasekaran P, Pappu HR (2023) Yeast two-hybrid technique to identify protein-protein interactions. Methods Mol Biol 2690: 1−8
Goodman JM (2008) The gregarious lipid droplet. J Biol Chem 283(42): 28005−28009
Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, Londos C (1991) Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J Biol Chem 266(17): 11341−11346
Greenberg AS, Egan JJ, Wek SA, Moos MC, Jr., Londos C, Kimmel AR (1993) Isolation of cDNAs for perilipins A and B: sequence and expression of lipid droplet-associated proteins of adipocytes. Proc Natl Acad Sci USA 90(24): 12035−12039
Jiang HP, Serrero G (1992) Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. Proc Natl Acad Sci USA 89(17): 7856−7860
Kalscheuer R, Waltermann M, Alvarez M, Steinbuchel A (2001) Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins. Arch Microbiol 177(1): 20−28
Kernohan EA, Lepherd EE (1969) Size distribution of fat globules in cow's milk during milking, measured with a Coulter counter. J Dairy Res 36(2): 177−182
Kim SY, Hakoshima T (2019) GST pull-down assay to measure complex formations. Methods Mol Biol 1893: 273−280
Lin JS, Lai EM (2017) Protein-protein interactions: co-immunoprecipitation. Methods Mol Biol 1615: 211−219
Liu P, Ying Y, Zhao Y, Mundy DI, Zhu M, Anderson RG (2004) Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J Biol Chem 279(5): 3787−3792
Lo Sardo F (2023) Co-immunoprecipitation (Co-Ip) in mammalian cells. Methods Mol Biol 2655: 67−77
Martin S, Parton RG (2006) Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7(5): 373−378
Masters SC (2004) Co-immunoprecipitation from transfected cells. Methods Mol Biol 261: 337−350
Miura S, Gan JW, Brzostowski J, Parisi MJ, Schultz CJ, Londos C, Oliver B, Kimmel AR (2002) Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J Biol Chem 277(35): 32253−32257
Murphy S, Martin S, Parton RG (2009) Lipid droplet-organelle interactions; sharing the fats. Biochim Biophys Acta 1791(6): 441−447
Ohsaki Y, Cheng J, Suzuki M, Shinohara Y, Fujita A, Fujimoto T (2009) Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure. Biochim Biophys Acta 1791(6): 399−407
Olzmann JA, Carvalho P (2019) Dynamics and functions of lipid droplets. Nat Rev Mol Cell Biol 20(3): 137−155
Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC, Hobbs HH (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 40(12): 1461−1465
Su W, Wang Y, Jia X, Wu W, Li L, Tian X, Li S, Wang C, Xu H, Cao J, Han Q, Xu S, Chen Y, Zhong Y, Zhang X, Liu P, Gustafsson JA, Guan Y (2014) Comparative proteomic study reveals 17beta-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease. Proc Natl Acad Sci USA 111(31): 11437−11442
Sztalryd C, Xu G, Dorward H, Tansey JT, Contreras JA, Kimmel AR, Londos C (2003) Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol 161(6): 1093−1103
Tan L, Yammani RR (2022) Co-immunoprecipitation-blotting: analysis of protein-protein interactions. Methods Mol Biol 2413: 145−154
Wan HC, Melo RC, Jin Z, Dvorak AM, Weller PF (2007) Roles and origins of leukocyte lipid bodies: proteomic and ultrastructural studies. FASEB J 21(1): 167−178
Wang H, Bell M, Sreenivasan U, Sreenevasan U, Hu H, Liu J, Dalen K, Londos C, Yamaguchi T, Rizzo MA, Coleman R, Gong D, Brasaemle D, Sztalryd C (2011) Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein. J Biol Chem 286(18): 15707−15715
Wang Y, Kory N, BasuRay S, Cohen JC, Hobbs HH (2019) PNPLA3, CGI-58, and inhibition of hepatic triglyceride hydrolysis in mice. Hepatology 69(6): 2427−2441
Xie K, Zhang P, Na H, Liu Y, Zhang H, Liu P (2019) MDT-28/PLIN-1 mediates lipid droplet-microtubule interaction via DLC-1 in Caenorhabditis elegans. Sci Rep 9(1): 14902. https://doi.org/10.1038/s41598-019-51399-z
Yang L, Ding Y, Chen Y, Zhang S, Huo C, Wang Y, Yu J, Zhang P, Na H, Zhang H, Ma Y, Liu P (2012) The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans. J Lipid Res 53(7): 1245−1253
Zehmer JK, Huang Y, Peng G, Pu J, Anderson RG, Liu P (2009) A role for lipid droplets in inter-membrane lipid traffic. Proteomics 9(4): 914−921
Zhang H, Wang Y, Li J, Yu J, Pu J, Li L, Zhang H, Zhang S, Peng G, Yang F, Liu P (2011) Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I. J Proteome Res 10(10): 4757−4768
Zhang P, Na H, Liu Z, Zhang S, Xue P, Chen Y, Pu J, Peng G, Huang X, Yang F, Xie Z, Xu T, Xu P, Ou G, Zhang SO, Liu P (2012) Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets. Mol Cell Proteomics 11(8): 317−328
Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R (2004) Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306(5700): 1383−1386
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