Volume 10 Issue 2
Apr.  2024
Turn off MathJax
Article Contents
Xiaochuan Fu, Shuyan Zhang, Pingsheng Liu. Co-immunoprecipitation for identifying protein–protein interaction on lipid droplets. Biophysics Reports, 2024, 10(2): 102-110. doi: 10.52601/bpr.2024.240007
Citation: Xiaochuan Fu, Shuyan Zhang, Pingsheng Liu. Co-immunoprecipitation for identifying protein–protein interaction on lipid droplets. Biophysics Reports, 2024, 10(2): 102-110. doi: 10.52601/bpr.2024.240007

Co-immunoprecipitation for identifying protein–protein interaction on lipid droplets

doi: 10.52601/bpr.2024.240007
More Information
  • Corresponding author: syzhang@ibp.ac.cn (S. Zhang); pliu@ibp.ac.cn (P. Liu)
  • Received Date: 27 January 2024
  • Accepted Date: 23 February 2024
  • Available Online: 19 March 2024
  • Publish Date: 30 April 2024
  • 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.

  • Xiaochuan Fu, Shuyan Zhang and Pingsheng Liu declare that they have no conflict of interest.
    All institutional and national guidelines for the care and use of laboratory animals were followed.

  • loading
  • 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 doi: 10.1056/NEJMoa1712191
    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 doi: 10.1128/JB.181.20.6441-6448.1999
    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 doi: 10.1021/pr070158j
    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 doi: 10.1074/mcp.M600011-MCP200
    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 doi: 10.1016/S0022-2275(20)34939-7
    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 doi: 10.3390/ijms10062763
    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 doi: 10.1194/jlr.M021899
    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 doi: 10.1038/nprot.2012.142
    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 doi: 10.1073/pnas.89.18.8537
    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 doi: 10.1016/j.cell.2009.11.005
    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 doi: 10.1007/s00418-008-0449-0
    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 doi: 10.1194/jlr.M700050-JLR200
    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 doi: 10.1016/j.bbamcr.2003.10.018
    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 doi: 10.1074/jbc.R800042200
    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 doi: 10.1016/S0021-9258(18)99168-4
    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 doi: 10.1073/pnas.90.24.12035
    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 doi: 10.1073/pnas.89.17.7856
    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 doi: 10.1007/s00203-001-0355-5
    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 doi: 10.1017/S002202990001267X
    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 doi: 10.1074/jbc.M311945200
    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 doi: 10.1038/nrm1912
    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 doi: 10.1074/jbc.M204410200
    Murphy S, Martin S, Parton RG (2009) Lipid droplet-organelle interactions; sharing the fats. Biochim Biophys Acta 1791(6): 441−447 doi: 10.1016/j.bbalip.2008.07.004
    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 doi: 10.1016/j.bbalip.2008.10.002
    Olzmann JA, Carvalho P (2019) Dynamics and functions of lipid droplets. Nat Rev Mol Cell Biol 20(3): 137−155 doi: 10.1038/s41580-018-0085-z
    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 doi: 10.1038/ng.257
    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 doi: 10.1073/pnas.1410741111
    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 doi: 10.1083/jcb.200210169
    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 doi: 10.1096/fj.06-6711com
    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 doi: 10.1074/jbc.M110.207779
    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 doi: 10.1002/hep.30583
    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
    Xu S, Zhang X, Liu P (2018) Lipid droplet proteins and metabolic diseases. Biochim Biophys Acta Mol Basis Dis 1864(5 Pt B): 1968-1983
    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 doi: 10.1194/jlr.R024117
    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 doi: 10.1002/pmic.200800584
    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 doi: 10.1021/pr200553c
    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 doi: 10.1074/mcp.M111.016345
    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 doi: 10.1126/science.1100747
  • 加载中


    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(2)  / Tables(2)

    Article Metrics

    Article views (425) PDF downloads(43) Cited by()
    Proportional views


    DownLoad:  Full-Size Img  PowerPoint