Synchronizing Protein Transport in the Secretory Pathway

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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)
Asc I, Eco RI, Fse I, and Pac I 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)
T₄ 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

Figure 15.19.1 Principle of the RUSH system. The RUSH (retention using selective hooks) system is a two‐state secretory assay. The reporter protein is retained in the donor compartment via interaction with the hook mediated by the streptavidin‐SBP (streptavidin‐binding peptide) interaction. Addition of biotin allows release of the reporter from the hook. The reporter then trafficks to its acceptor compartment in a synchronous way. To allow an easier observation, the reporter is fused to a fluorescent protein.

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Figure 15.19.2 Model of a RUSH plasmid and examples of reporter protein topologies. (A ) The hook is placed upstream of the IRES (internal ribosome entry site) while the reporter is downstream of it. The four cloning cassettes (A to D) enable cloning of the different domains necessary to create a reporter fusion protein. They are separated by the indicated restriction sites. (B ) The topology of the protein of interest and the orientation of the retention decipher the location of the reporter domains to be cloned in cassettes A to D. Abbreviations: FP, fluorescent protein; IL2ss, Interleukin‐2 signal sequence; SBP, streptavidin‐binding peptide; X, protein of interest.

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Figure 15.19.3 Release of the Golgi enzyme alpha‐(1, 6)‐fucosyltransferase (FUT8‐SBP‐EGFP). Coverslips were prepared as indicated in . The plasmid encoding for the alpha‐(1, 6)‐fucosyltransferase as a reporter was constructed as described in . The FUT8 gene was inserted between Asc I and Eco RI sites, and since the alpha‐(1, 6)‐fucosyltransferase is a type II transmembrane protein, the retention occurs in the lumen using Str‐KDEL as a hook. HeLa cells were transiently transfected with a plasmid encoding for Str‐KDEL and FUT8‐SBP‐EGFP. Release of the reporter was induced by the addition of biotin in the medium, and coverslips were fixed after 15 min or 60 min of incubation. The nontreated coverslip shows the retention state, and the coverslip incubated with biotin since the beginning of transfection (steady‐state) shows the steady‐state behavior of the reporter in the absence of retention. Scale bar: 10 µm.

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Figure 15.19.4 Release of the plasma membrane–fated SBP‐EGFP‐GPI. Coverslips were prepared as indicated in the . The plasmid encoding for the EGFP fused to a glycosylphosphatidylinositol (GPI) anchor as a reporter was constructed as described in . The Interleukin‐2 signal peptide was inserted between Asc I and Eco RI and the GPI anchor between Fse I and Pac I sites. The retention occurs in the lumen using Str‐KDEL as a hook. HeLa cells were transiently transfected with a plasmid encoding for Str‐KDEL and SBP‐EGFP‐GPI. Release of the reporter was induced by the addition of biotin in the medium and coverslips were fixed after 15 min or 60 min of incubation. The nontreated coverslip shows the retention state and the coverslip incubated with biotin since the beginning of transfection (steady‐state) shows the steady‐state behavior of the reporter in the absence of retention. Scale bar: 10 µm.

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Figure 15.19.5 Schemes of the Chamlide for release under the microscope. (A ) Components of the L‐shaped Chamlide. (B ) Assembled L‐shaped Chamlide with tubing and syringes to replace medium for real‐time release of the reporter. Pictures adapted from www.chamlide.com (Live Cell instruments).

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Figure 15.19.6 Schematic diagram showing results that could be obtained for release of a soluble secretory protein to the extracellular medium. (A ) Localization of the secretory protein after different incubation times with biotin. At 0 min, the protein is retained in the endoplasmic reticulum. At 10 min, it is located in the Golgi complex and after 60 min it is released in the extracellular medium and disappears from the intracellular space. (B ) At time 0 min, the protein is detected only in cell lysates since it is retained in the endoplasmic reticulum. At 10 min, the protein is still mainly detected in the cell lysates. Depending on the kinetics of the reporter, some protein may already be detected in the concentrated supernatants. After 60 min of incubation in the presence of biotin, the protein was released from the cell and is mainly detected in the concentrated supernatants confirming it was secreted out of the cells. Abbreviation: NT, nontransfected cells.

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Literature Cited

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