In Vitro Analysis of the Very‐Low Density Lipoprotein Export from the Trans‐Golgi Network

Shadab A. Siddiqi1

1 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.21
DOI:  10.1002/0471143030.cb1121s67
Online Posting Date:  June, 2015
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Abstract

The movement of mature VLDL particles from the TGN to the plasma membrane (PM) is a complex physiological process that plays a critical role in hepatic lipid homeostasis. However, the molecular mechanisms regulating these intracellular transport events had not been studied until recently because of the lack of appropriate molecular assays and techniques. This unit provides a detailed description of cell‐free approaches and techniques to study the TGN‐to‐PM transport of the mature VLDL at the molecular level. A major emphasis is placed on the preparation and purification of sub‐cellular organelles because the success of in vitro assays for the vesicle formation and fusion depends on the quality of the isolated TGN, hepatic PM and hepatic cytosol. A number of critical factors that control the formation of mature VLDL‐containing vesicle, the PG‐VTV, from the TGN and their subsequent targeting to and fusion with the hepatic PM have been discussed. © 2015 by John Wiley & Sons, Inc.

Keywords: trans‐Golgi network (TGN); very low‐density lipoprotein (VLDL); post‐golgi VLDL transport vesicle (PG‐VTV); apolipoprotein B; endoplasmic reticulum; triacylglycerol

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro TGN Budding and Isolation of TGN‐Derived Mature VLDL Containing Vesicles
  • Basic Protocol 2: In Vitro Fusion of PG‐VTV with Plasma Membranes
  • Support Protocol 1: Isolation and Purification of Rat Hepatic TGN Membranes Containing [3H]TAG
  • Support Protocol 2: Preparation of Rat Hepatic Cytosol
  • Support Protocol 3: Isolation and Purification of Rat Hepatic Plasma Membrane
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro TGN Budding and Isolation of TGN‐Derived Mature VLDL Containing Vesicles

  Materials
  • Rat hepatic TGN membranes containing [3H]TAG; 200 μg (see protocol 3)
  • Rat hepatic cytosol; 500 μg (see protocol 4)
  • Ice and ice bucket
  • 10 mM HEPES, pH 7.2
  • 0.1 M and 0.86 M sucrose solutions in 10 mM HEPES
  • 80:20:2 (v/v) Isopropanol/n‐heptane/deionized water solution
  • n‐heptane
  • 0.5 N 1:5:5 (v/v) NaOH/absolute ethyl alcohol/deionized water solution
  • Liquid nitrogen
  • 50 mM diethyl‐p‐nitrophenylphosphate (E600)
  • Water bath at 37°C
  • 5‐ml glass tubes
  • 12‐ml polyallomer centrifuge tube (Beckman)
  • Centrifuge (Beckman SW41 Ti rotor)
  • Pasteur pipets
  • Vortex mixer
  • Liquid scintillation counter
NOTE: All addition steps are carried out on ice using an ice bucket with prechilled buffers.

Basic Protocol 2: In Vitro Fusion of PG‐VTV with Plasma Membranes

  Materials
  • PG‐VTV membranes containing [3H]TAG: 150 μg (see protocol 1)
  • Rat hepatic cytosol: 500 μg (see protocol 4)
  • Rat hepatic plasma membranes (PM): 150 μg (see Basic Protocol 3)
  • Ice and ice bucket
  • Sucrose solutions (0.69 M, 0.9 M, and 1.12 M) in 10 mM HEPES
  • 80:20:2 (v/v) Isopropanol/n‐heptane/deionized water solution
  • n‐heptane
  • 0.5 N NaOH/absolute ethyl alcohol/deionized water solution (1/5/5; v/v)
  • Water bath at 37°C
  • 5‐ml glass tubes
  • 12‐ml polyallomer centrifuge tubes (Beckman)
  • Centrifuge (with a Beckman SW41 Ti rotor)
  • Pasteur pipets
  • Vortex mixer
  • Liquid scintillation counter
NOTE: All additional steps are carried out on ice using ice bucket with pre‐chilled buffers.

Support Protocol 1: Isolation and Purification of Rat Hepatic TGN Membranes Containing [3H]TAG

  Materials
  • Primary rat hepatocytes, freshly isolated from one Sprague‐Dawley rat (150 to 200 g; see Tiwari et al., )
  • Buffer A (see recipe)
  • Ice and ice bucket
  • BSA‐oleic acid complex (Sigma‐Aldrich, cat. no. O3008‐5 ML)
  • [3H]oleic acid (45.5.Ci/mM) (PerkinElmer Life Sciences)
  • 2% (w/v) BSA in phosphate‐buffered saline (PBS; see recipe)
  • Buffer B (see recipe)
  • Protease inhibitors cocktail (Roche Applied Science, cat. no. 04693116001)
  • Sucrose solutions (0.25 M, 0.86 M, 1.15 M, and 2.1 M) in 10 mM HEPES
  • Liquid nitrogen
  • Bradford assay
  • Transport buffer (see recipe)
  • Vortex mixer
  • Water bath at 37°C
  • Parr cell disruption vessel (Parr Instruments; Model 4635)
  • 30‐ml centrifuge tubes
  • Sorvall centrifuge using Fiberlite F21S‐8 × 50 y rotor (Thermo Scientific)
  • Thick‐walled polycarbonate centrifuge bottle with caps (Beckman) for Beckman 70 Ti rotor
  • Beckman 70 Ti rotor
  • 12‐ml polyallomer centrifuge tubes (Beckman)
  • Pasteur pipets

Support Protocol 2: Preparation of Rat Hepatic Cytosol

  Materials
  • Primary rat hepatocytes, freshly isolated from one to two Sprague‐Dawley rat (150 to 200 g; see Tiwari et al., )
  • Ice and ice bucket
  • Buffer C (see recipe)
  • Protease inhibitors cocktail (Roche Applied Science, cat. no. 04693116001)
  • Cytosol
  • Liquid nitrogen
  • Transport buffer (see recipe)
  • Parr cell disruption vessel (Parr Instruments; Model 4635)
  • 30‐ml centrifuge tube
  • Thick‐walled polycarbonate centrifuge bottle with cap for Beckman 70 Ti rotor
  • Sorvall centrifuge using Fiberlite F21S‐8 × 50 y rotor (Thermo Scientific)
  • 10 kDa MWCO membrane (Spectra/Por)
  • Beckman 70 Ti rotor
  • Glass Pasteur pipets
  • Amicon stirred cell (Millipore, model 8200)
  • Centricon tubes (YM‐10 membrane)

Support Protocol 3: Isolation and Purification of Rat Hepatic Plasma Membrane

  Materials
  • Freshly harvested rat liver from one Sprague‐Dawley rat (150 to 200 g)
  • Ice and ice bucket
  • Buffer B (see recipe)
  • Protease inhibitors cocktail (Roche Applied Science, cat. no. 04693116001)
  • Sucrose solutions (0.69 M, 0.9 M, and 1.12 M) in 10 mM HEPES
  • 10 mM HEPES, pH 7.2
  • Liquid nitrogen
  • Clean scalpel
  • Potter‐Elvehjem tissue homogenizer with polytetrafluorethylene pestle (Corning, cat. no. 7725 T‐8)
  • Parr cell disruption vessel (Parr Instruments, model 4635)
  • 30‐ml thick‐walled polycarbonate centrifuge tubes with caps for Beckman 70 Ti rotor
  • Sorvall centrifuge using Fiberlite F21S‐8 × 50 y rotor (Thermo Scientific)
  • Fiberlite F21S‐8 × 50 y rotor (Thermo Scientific)
  • 12‐ml polyallomer centrifuge tubes (Beckman)
  • Beckman SW41 Ti rotor
  • Glass Pasteur pipets
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Figures

Videos

Literature Cited

Literature Cited
  Fisher, E.A. and Ginsberg, H.N. 2002. Complexity in the secretory pathway: The assembly and secretion of apolipoprotein B‐containing lipoproteins. J. Biol. Chem. 277:17377‐17380.
  Gibbons, G.F., Khurana, R., Odwell, A., and Seelaender, M.C. 1994. Lipid balance in HepG2 cells: Active synthesis and impaired mobilization. J. Lipid. Res. 35:1801‐1808.
  Ginsberg, H.N. 1995. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr. Opin. Lipidol. 6:275‐280.
  Graham, J.M. 2001. Isolation of Golgi membranes from tissues and cells by differential and density gradient centrifugation. Curr. Protoc. Cell Biol. 10:3.9.1‐3.9.24.
  Gusarova, V., Seo, J., Sullivan, M.L., Watkins, S.C., Brodsky, J.L., and Fisher, E.A. 2007. Golgi‐associated maturation of very low density lipoproteins involves conformational changes in apolipoprotein B, but is not dependent on apolipoprotein E. J. Biol. Chem. 282:19453‐19462.
  Hossain, T., Riad, A., Siddiqi, S., Parthasarathy, S., and Siddiqi, S.A. 2014. Mature VLDL triggers the biogenesis of a distinct vesicle from the trans‐Golgi network for its export to the plasma membrane. Biochem. J. 459:47‐58.
  Hussain, M.M., Shi, J., and Dreizen, P. 2003. Microsomal triglyceride transfer protein and its role in apoB‐lipoprotein assembly. J. Lipid Res. 44:22‐32.
  Hussain, M.M., Rava, P., Pan, X., Dai, K., Dougan, S.K., Iqbal, J., Lazare, F., and Khatun, I. 2008. Microsomal triglyceride transfer protein in plasma and cellular lipid metabolism. Curr. Opin. Lipidol. 19:277‐284.
  Mahmood, T. and Yang, P‐C. 2012. Western blot: Technique, theory, and trouble shooting. N. Am. J. Med. Sci. 4:429‐434.
  Mansbach, C.M. and Siddiqi, S.A. 2010. The biogenesis of chylomicrons. Annu. Rev. Physiol. 72:315‐333.
  Olofsson, S.O. and Boren, J. 2005. Apolipoprotein B: A clinically important apolipoprotein which assembles atherogenic lipoproteins and promotes the development of atherosclerosis. J. Intern. Med. 258:395‐410.
  Olofsson, S.O., Asp, L., and Boren, J. 1999. The assembly and secretion of apolipoprotein B‐containing lipoproteins. Curr. Opin. Lipidol. 10:341‐346.
  Palmgren, M.G., Askerlund, P., Fredrikson, K., Widell, S., Sommarin, M., and Larsson, C. 1990. Sealed inside‐out and right‐side‐out plasma membrane vesicles : Optimal conditions for formation and separation. Plant Physiol. 92:871‐880.
  Rahim, A., Nafi‐Valencia, E., Siddiqi, S., Basha, R., Runyon, C.C., and Siddiqi, S.A. 2012. Proteomic analysis of the very low density lipoprotein (VLDL) transport vesicles. J. Proteomics 75:2225‐2235.
  Shelness, G.S., Ingram, M.F., Huang, X.F., and Delozier, J.A. 1999. Apolipoprotein B in the rough endoplasmic reticulum: Translation, translocation and the initiation of lipoprotein assembly. J. Nutr. 129:456S‐462S.
  Siddiqi, S.A. 2008. VLDL exits from the endoplasmic reticulum in a specialized vesicle, the VLDL transport vesicle, in rat primary hepatocytes. Biochem. J. 413:333‐342.
  Siddiqi, S.A., Gorelick, F.S., Mahan, J.T., and Mansbach, C.M., 2nd. 2003. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre‐chylomicron transport vesicle. J. Cell Sci. 116:415‐427.
  Siddiqi, S.A., Mahan, J., Siddiqi, S., Gorelick, F.S., and Mansbach, C.M. 2nd. 2006a. Vesicle‐associated membrane protein 7 is expressed in intestinal ER. J. Cell Sci. 119:943‐950.
  Siddiqi, S.A., Siddiqi, S., Mahan, J., Peggs, K., Gorelick, F.S., and Mansbach, C.M., 2nd. 2006b. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle. J. Biol. Chem. 281:20974‐20982.
  Siddiqi, S., Mani, A.M., and Siddiqi, S.A. 2010a. The identification of the SNARE complex required for the fusion of VLDL‐transport vesicle with hepatic cis‐Golgi. Biochem. J. 429:391‐401.
  Siddiqi, S., Saleem, U., Abumrad, N.A., Davidson, N.O., Storch, J., Siddiqi, S.A., and Mansbach, C.M., 2nd. 2010b. A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER. J. Lipid Res. 51:1918‐1928.
  Tiwari, S. and Siddiqi, S.A. 2012. Intracellular trafficking and secretion of VLDL. Arterioscler Thromb. Vasc. Biol. 32:1079‐1086.
  Tiwari, S., Siddiqi, S., and Siddiqi, S.A. 2013. CideB protein is required for the biogenesis of very low density lipoprotein (VLDL) transport vesicle. J. Biol. Chem. 288:5157‐5165.
  Tran, K., Thorne‐Tjomsland, G., Delong, C.J., Cui, Z., Shan, J., Burton, L., Jamieson, J.C., and Yao, Z. 2002. Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA‐RH7777 cells. J. Biol. Chem. 277:31187‐31200.
  Tsai, J., Qiu, W., Kohen‐Avramoglu, R., and Adeli, K. 2007. MEK‐ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: Potential role of ERK in VLDL‐ ApoB100 particle assembly. Arterioscler. Thromb. Vasc. Biol. 27:211‐218.
  Yao, Z. and Mcleod, R.S. 1994. Synthesis and secretion of hepatic apolipoprotein B‐containing lipoproteins. Biochim. Biophys. Acta 1212:152‐166.
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