Synchronizing Protein Transport in the Secretory Pathway

Gaelle Boncompain1, Franck Perez1

1 CNRS UMR144, Paris, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.19
DOI:  10.1002/0471143030.cb1519s57
Online Posting Date:  December, 2012
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Abstract

To be secreted or transported to their target compartments, newly synthesized proteins leave the endoplasmic reticulum to reach the Golgi apparatus, where they are processed and sorted toward their final destinations along the secretory pathway. It is now clear that many Golgi‐intersecting and non‐intersecting pathways exist in cells to carry out proper transport, modification, and addressing. To analyze and visualize the intracellular trafficking of any secretory protein, we developed the retention using selective hooks (RUSH) system. This assay allows the simultaneous release of a pool of particular secretory proteins from the endoplasmic reticulum and the monitoring of their anterograde trafficking. The use of the RUSH system is detailed in these protocols, from sub‐cloning the sequence coding for the protein of interest into RUSH plasmids to visualization of its trafficking. Curr. Protoc. Cell Biol. 57:15.19.1‐15.19.16. © 2012 by John Wiley & Sons, Inc.

Keywords: intracellular trafficking; secretory pathway

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning the Reporter of Interest into the RUSH Plasmid
  • Basic Protocol 2: Test the Retention and Release of the Protein of Interest in the RUSH System on Fixed Samples
  • Support Protocol 1: Test the Arrival at the Plasma Membrane of a Plasma Membrane‐Fated Reporter
  • Basic Protocol 3: Follow Trafficking of the Reporter Protein in Living Cells
  • Alternate Protocol 1: Monitor Release of a Secretory Soluble Protein in the Culture Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cloning the Reporter of Interest into the RUSH Plasmid

  Materials
  • RUSH vectors (Boncompain et al., )
  • NEB buffer 4 (New England Biolabs)
  • AscI, EcoRI, FseI, and PacI enzymes (New England Biolabs or equivalent)
  • Alkaline phosphatase and 10× alkaline phosphatase buffer
  • Agarose gel
  • NucleoSpin Gel and PCR Clean‐up kit (Macherey‐Nagel or equivalent)
  • T 4 DNA ligase and 10× ligation buffer
  • Gene of interest flanked with convenient restriction sites (amplified by PCR or as a synthetic DNA)
  • E. coli thermocompetent cells (e.g., DG1 from Eurogentec)
  • LB agar plates with ampicillin
  • NucleoSpin Xtra Midi Plus kit (Macherey‐Nagel)

Basic Protocol 2: Test the Retention and Release of the Protein of Interest in the RUSH System on Fixed Samples

  Materials
  • Adherent cells
  • RUSH plasmid coding for the protein of interest as a reporter (see protocol 1)
  • Biotin, 4 mM stock solution (see recipe)
  • Culture medium
  • 3% paraformaldehyde (PFA) solution in PBS [prepared from a commercial 16% PFA solution (Electron Microscopy Sciences, cat. no. 15710) diluted in PBS]
  • Phosphate‐buffered saline (PBS)
  • Quenching solution (see recipe)
  • 10× Permeabilizing solution (see recipe)
  • Primary antibody against streptavidin (e.g., monoclonal anti‐Streptavidin clone S10D4)
  • Secondary antibody coupled to a fluorophore
  • Mounting medium with DAPI
  • 12‐mm glass coverslips
  • Parafilm
  • Humidified chamber
  • Glass slides
  • Epifluorescence or confocal microscope

Support Protocol 1: Test the Arrival at the Plasma Membrane of a Plasma Membrane‐Fated Reporter

  • Primary antibody directed to an external domain of the reporter or to the EGFP if extracellular
  • Phosphate‐buffered saline (PBS), ice‐cold
  • Ice
  • Metal plate
  • 24‐well plates

Basic Protocol 3: Follow Trafficking of the Reporter Protein in Living Cells

  Materials
  • Adherent cells
  • Culture medium
  • RUSH plasmid coding for the protein of interest as a reporter (see protocol 1)
  • Leibovitz's medium
  • 4 mM biotin stock solution (see recipe)
  • Immersion oil
  • 25‐mm glass coverslips and incubation chamber for these coverslips (e.g., L‐shaped tubing Chamlide)
  • 1‐ and 5‐ml syringes
  • Plastic tubing
  • Spinning disk microscope with an inverted objective and a thermostated incubation chamber

Alternate Protocol 1: Monitor Release of a Secretory Soluble Protein in the Culture Medium

  Materials
  • Adherent cells
  • RUSH plasmid coding for the protein of interest as a reporter (see protocol 1)
  • Culture medium without serum
  • 4 mM biotin stock solution (see recipe)
  • 2× Laemmli buffer containing 4% 2‐mercaptoethanol (see recipe)
  • 1× Laemmli buffer containing 2% 2‐mercaptoethanol (see recipe)
  • Polyacrylamide gel (unit 6.1)
  • 10‐cm petri dishes
  • Plastic cell scraper
  • Plastic tubes with cap
  • Hemacytometer
  • Centrifuge
  • Vivaspin protein concentrator
  • Pipet tips
  • SDS‐PAGE electrophoresis equipment
  • Immunoblot and immunodetection equipment
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Figures

Videos

Literature Cited

Literature Cited
   Bard, F., Casano, L., Mallabiabarrena, A., Wallace, E., Saito, K., Kitayama, H., Guizzunti, G., Hu, Y., Wendler, F., Dasgupta, R., Perrimon, N., and Malhotra, V. 2006. Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439:604‐607.
   Boncompain, G., Divoux, S., Gareil, N., de Forges, H., Lescure, A., Latreche, L., Mercanti, V., Jollivet, F., Raposo, G., and Perez, F. 2012. Synchronization of secretory protein traffic in populations of cells. Nat. Methods 9:493‐498.
   D'Angelo, G., Prencipe, L., Iodice, L., Beznoussenko, G., Savarese, M., Marra, P., Di Tullio, G., Martire, G., De Matteis, M.A., and Bonatti, S. 2009. GRASP65 and GRASP55 sequentially promote the transport of C‐terminal valine‐bearing cargos to and through the Golgi complex. J. Biol. Chem. 284:34849‐34860.
   Gordon, D.E., Bond, L.M., Sahlender, D.A., and Peden, A.A. 2010. A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells. Traffic 11:1191‐1204.
   Hicks, S.W., Horn, T.A., McCaffery, J.M., Zuckerman, D.M., and Machamer, C.E. 2006. Golgin‐160 promotes cell surface expression of the beta‐1 adrenergic receptor. Traffic 7:1666‐1677.
   Kondylis, V., Tang, Y., Fuchs, F., Boutros, M., and Rabouille, C. 2011. Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen.PLoS One 6:e17173.
   Kreis, T.E. and Lodish, H.F. 1986. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell 46:929‐937.
   Lafay, F. 1974. Envelope proteins of vesicular stomatitis virus: effect of temperature‐sensitive mutations in complementation groups III and V. J. Virol. 14:1220‐1228.
   Lieu, Z.Z., Lock, J.G., Hammond, L.A., La Gruta, N.L., Stow, J.L., and Gleeson, P.A. 2008. A trans‐Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:3351‐3356.
   Lippincott‐Schwartz, J., Yuan, L.C., Bonifacino, J.S., and Klausner, R.D. 1989. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: Evidence for membrane cycling from Golgi to ER. Cell 56:801‐813.
   Lock, J.G., Hammond, L.A., Houghton, F., Gleeson, P.A., and Stow, J.L. 2005. E‐cadherin transport from the trans‐Golgi network in tubulovesicular carriers is selectively regulated by golgin‐97. Traffic 6:1142‐1156.
   Matlin, K.S. and Simons, K. 1983. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34:233‐243.
   Presley, J.F., Cole, N.B., Schroer, T.A., Hirschberg, K., Zaal, K.J., and Lippincott‐Schwartz, J. 1997. ER‐to‐Golgi transport visualized in living cells. Nature 389:81‐85.
   Rivera, V.M., Wang, X., Wardwell, S., Courage, N.L., Volchuk, A., Keenan, T., Holt, D.A., Gilman, M., Orci, L., Cerasoli, F. Jr., Rothman, J.E., and Clackson, T. 2000. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science 287:826‐830.
   Saraste, J. and Kuismanen, E. 1984. Pre‐ and post‐Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell 38:535‐549.
   Scales, S.J., Pepperkok, R., and Kreis, T.E. 1997. Visualization of ER‐to‐Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90:1137‐1148.
   Simpson, J.C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.G., Singan, V.R., Heriche, J.K., Neumann, B., Mateos, A., Blake, J., Bechtel, S., Benes, V., Wiemann, S., Ellenberg, J., and Pepperkok, R. 2012. Genome‐wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway. Nat. Cell Biol. 14:764‐774.
   Thomason, L., Court, D.L., Bubunetko, M., Costantino, N., Wilson, H., Datta, S., and Oppenheim, A. 2007. Recombineering: Genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 78:1.16.1‐1.16.24.
   Wendler, F., Gillingham, A.K., Sinka, R., Rosa‐Ferreira, C., Gordon, D.E., Franch‐Marro, X., Peden, A.A., Vincent, J.P., and Munro, S. 2010. A genome‐wide RNA interference screen identifies two novel components of the metazoan secretory pathway. EMBO J. 29:304‐314.
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