Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Summary

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Abstract

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (<30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Introduction

The folding of even relatively simple proteins involves many different folding enzymes, molecular chaperones, and covalent modifications¹. A complete reconstitution of these processes in vitro is practically impossible, given the vast number of different components involved. It is highly desirable, therefore, to study protein folding in vivo, in live cells. Radioactive pulse-chase techniques prove a powerful tool for studying the synthesis, folding, transport, and degradation of proteins in their natural environment.

The metabolic labeling of proteins during a short pulse with ³⁵S-labeled methionine/cysteine, followed by a chase in the absence of a radioactive label, allows specific tracking of a population of newly synthesized proteins in the wider cellular milieu. Then, target proteins can be isolated via immunoprecipitation and analyzed via SDS-PAGE or other techniques. For many proteins, their journey through the cell is marked by modifications that are visible on SDS-PAGE gel. For example, the transport of glycosylated proteins from the endoplasmic reticulum (ER) to the Golgi complex is often accompanied by modifications of N-linked glycans or the addition of O-linked glycans^2,3. These modifications cause large increases in the apparent molecular mass, which can be seen by mobility changes in SDS-PAGE. Maturation can also be marked by proteolytic cleavages, such as signal-peptide cleavage or the removal of pro-peptides, resulting in changes in the apparent molecular mass that can be followed easily on SDS-PAGE gel⁴. Radioactivity has considerable advantages over comparable techniques such as cycloheximide chases, where novel protein synthesis is prevented, as longer treatments are toxic to cells and do not exclude the majority of older, steady-state proteins from the analysis, as some proteins have half-lives of days. The comparison of proteins under both nonreducing and reducing conditions allows the analysis of disulfide bond formation, an important step in the folding of many secretory proteins^4,5,6,7.

Here we describe a general method for the analysis of protein folding and transport in intact cells, using a radioactive pulse-chase approach. While we have aimed to provide the method as detailed as possible, the protocol has an almost limitless potential for adaptability and will allow optimization to study each reader's specific proteins.

Two alternative pulse-chase protocols, one for adherent cells (step 1.1 of the protocol presented here) and one for suspension cells (step 1.2 of the protocol presented here) are provided. The conditions provided here are sufficient to visualize a protein expressed with medium- to high-expression levels. If the reader is working with poorly expressed proteins or various posttreatment conditions, such as multiple immunoprecipitations, it is necessary to increase the dish size or cell number appropriately.

For suspension pulse chase, the chase samples taken at each time point are all taken from a single tube of cells. The wash steps after the pulse are omitted; instead, further incorporation of ³⁵S is prevented by dilution with a high excess of unlabeled methionine and cysteine.

The presented protocols use radioactive ³⁵S-labeled cysteine and methionine to follow cellular protein-folding processes. All operations with radioactive reagents should be performed using appropriate protective measures to minimize any exposure of the operator and the environment to radioactive radiation and be performed in a designated laboratory. As the pulse-chase labeling technique is relatively inefficient at short pulse times (<15 min), less than 1% of the starting amount of radioactivity is incorporated in the newly synthesized proteins. After the enrichment of the target protein via immunoprecipitation, the sample for SDS-PAGE contains less than 0.05% of the starting amount of radioactivity.

Although the ³⁵S methionine and cysteine labeling mix is stabilized, some decomposition, yielding volatile radioactive compounds, will occur. To protect the researcher and the apparatus, some precautions should be taken. The researcher should always obey the local radiation safety rules and may wear a charcoal nursing mask, besides a lab coat and (double) gloves. Stock vials with ³⁵S methionine and cysteine should always be opened in a fume hood, or under a local aspiration point. Known laboratory contamination spots are centrifuges, pipettes, water baths, incubators, and shakers. The contamination of these areas is reduced by using pipette tips with a charcoal filter, positive-seal microcentrifuge tubes (see Table of Materials), aquarium charcoal sponges in water baths, charcoal filter papers glued in the pulse-chase dishes, charcoal guard in the aspiration system, and the placement of dishes containing charcoal grains in incubators and storage containers.

Protocol

All radioactive reagents and procedures were handled in accordance with local Utrecht University radiation rules and regulations.

1. Pulse Chase

1. Pulse Chase for Adherent Cells
   NOTE: The volumes given here are based on 60 mm cell culture dishes. For 35 mm or 100 mm dishes, multiply the volumes by 1/2 or 2, respectively. This protocol uses a pulse time of 10 min and chase times of 0, 15, 30, 60, 120, and 240 min. These can be varied depending on the specific proteins being studied (discussed below).
   1. Seed adherent cells (e.g., HEK 293, HeLa, CHO) in six 60 mm cell culture dishes so that they will be subconfluent (80% - 90%) on the day of the pulse chase. Use at least one dish per time point and/or condition.
   2. If required, transfect the cells with commercially available transfection reagents according to the manufacturer's instructions; or, virally transduce⁸ the cells 1 day before the pulse-chase experiment with the appropriate construct for expression.
   3. Wash the dishes with 2 mL of wash buffer (Hank's balanced salt solution [HBSS]), add 2 mL of starvation medium (normal culture medium lacking methionine and cysteine and fetal bovine serum [FBS], such as minimum essential medium [MEM] without methionine and cysteine but with 10 mM HEPES, pH 7.4), and place the dishes in a 37 °C humidified incubator with 5% CO₂ for 15 min.
   4. Transfer the dishes to the racks in a prewarmed 37 °C water bath so that they are in contact with water but do not float. Start a timer.
   5. At 40 s, aspirate the starvation medium, draw up 600 µL of pulse solution (starvation media + 55 µCi/35 mm dish label, see the note preceding step 1.1.1) into the pipette, and add it gently to the center of the dish at exactly 1 min. Repeat this step at 1 min intervals for the remaining dishes. Start with the longest chase sample to save time during your experiment.
      NOTE: When handling radioactive material, it is essential to follow appropriate precautions and local rules and regulations to prevent accidental exposure and/or contamination.
   6. At exactly 11 min and for all following dishes, except for the 0 min chase sample, add 2 mL of chase medium directly to the dish, aspirate it, and again, add 2 mL of chase medium. Repeat this step at 1 min intervals for remaining dishes. Transfer all dishes to a 37 °C incubator.
   7. At exactly 16 min, add 2 mL of chase medium (normal culture medium + 10 mM HEPES [pH 7.4], 5 mM cysteine, and 5 mM methionine) directly to the 0 min sample dish on top of the pulse medium to stop labeling; then, aspirate immediately, transfer the dish to a cooled aluminum plate, and add 2 mL of ice-cold stop buffer (HBSS + 20 mM N-ethylmaleimide [NEM]).
      NOTE: This is the 0 min chase sample. For this sample, proceed directly to step 1.1.9.
   8. Transfer each chase dish back to the water bath 2 min before each chase time (e.g., 24 min for a 15 min chase) and, at the exact chase time (e.g., 26 min for a 15 min chase), aspirate the chase media (or transfer it to a microcentrifuge tube if following protein secretion) and transfer the dish to a cooled aluminum plate. Add 2 mL of ice-cold stop buffer.
   9. Incubate all dishes on ice in the stop buffer for ≥5 min; then, aspirate the stop solution and wash it with 2 mL of ice-cold stop solution. Aspirate the wash and lyse dishes with 600 µL of lysis buffer (phosphate-buffered saline [PBS] + nondenaturing detergent [see Table of Materials] + protease inhibitors + 20 mM NEM). Use a cell scraper to ensure that the dishes are lysed quantitatively.
   10. Transfer the lysate to a 1.5 mL microcentrifuge tube and centrifuge for 10 min at 15,000 - 20,000 x g and 4 °C to pellet the nuclei.
2. Pulse Chase for Suspension Cells
   NOTE: To ensure efficient labeling, cells should be pulsed at a concentration of 3 x 10⁶ to 5 x 10⁶ cells/mL, and chase volumes should be 4x the pulse volume. In the following example, we pulsed 5 x 10⁶ cells in a volume of 1 mL for 10 min, to yield five chase time points (0, 15, 30, 60, and 120 min) of 1 mL, containing 1 x 10⁶ cells per time point. All solutions are the same as in section 1.1.
   1. Culture the suspension cells (e.g., 3T3, Jurkat) according to a previously published protocol⁹ so that there is a sufficient number of cells at the time of the experiment, at least 1 x 10⁶ cells per time point and/or condition.
   2. If required, transfect cells with commercially available transfection reagents according to the manufacturer's instructions, or virally transduce⁸ the cells 1 day before the pulse-chase experiment with the appropriate construct for expression.
   3. Pellet 5 x 10⁶ cells per condition in a 50 mL tube for 5 min at 250 x g at room temperature, wash them 1x with 5 mL of starvation media, pellet them again, and resuspend them in 1 mL of starvation media.
   4. Transfer the cells to a 37 °C water bath and incubate them for 10 - 25 min. Agitate the tubes every 10 - 15 min to prevent the cells from settling at the bottom.
   5. Start the timer. At exactly 1 min, add 275 µCi (55 µCi/1 x 10⁶ cells) of undiluted label directly to the tube containing cells and swirl to mix.
      NOTE: When handling radioactive material, it is essential to follow appropriate precautions and local rules and regulations to prevent accidental exposure and/or contamination.
   6. At exactly 11 min, stop labeling by adding 4 mL of chase media. Mix the sample and immediately transfer 1 mL to a 15 mL tube on ice, containing 9 mL of ice-cold stop solution.
      NOTE: This is the 0 min chase sample.
   7. Repeat these steps for each successive time point. Once all time points are collected, pellet the cells for 5 min at 250 x g at 4 °C. Aspirate the medium (or transfer it to a new 15 mL tube if following protein secretion). Wash the cells with 5 mL of stop solution and pellet the cells for 5 min at 250 x g at 4 °C.
   8. Aspirate the stop solution, completely lyse the cells with 300 µL of ice-cold lysis buffer and incubate them for 20 min on ice to ensure a complete lysis. Transfer the lysate to a 1.5 mL microcentrifuge tube and centrifuge for 10 min at 15,000 - 20,000 x g at 4 °C to pellet the nuclei.

2. Immunoprecipitation

1. Combine antibody (see the discussion) and 50 µL of immunoprecipitation beads (e.g., protein A-Sepharose) (10% suspension [v/v] in lysis buffer + 0.25% bovine serum albumin [BSA]) in a microcentrifuge tube and incubate them at 4 °C for ~30 min in a shaker.
2. Pellet the beads for 1 min at 12,000 x g at room temperature and aspirate the supernatant. Add 200 µL of lysate to the antibody-bead mixture and incubate at 4 °C in a shaker for 1 h or head-over-head if the immunoprecipitation requires >1 h.
3. Pellet the beads for 1 min at 12,000 x g at room temperature. Aspirate the supernatant and add 1 mL of immunoprecipitation wash buffer. Place the sample in a shaker at room temperature for 5 min.
4. Pellet the beads as described in step 2.3 and repeat the wash 1x. Then, aspirate the supernatant and resuspend the beads in 20 µL of TE buffer, pH 6.8 (10 mM Tris-HCl, 1 mM EDTA). Vortex the sample.
5. Add 20 µL of 2x sample buffer without the reducing agent, vortex it, heat it for 5 min at 95 °C, and vortex again.
   NOTE: If only preparing reducing samples, 2 µL of 500 mM DTT should be added at this point. Then, proceed to step 2.7.
6. Pellet the beads as described in step 2.3. Transfer 19 µL of the nonreduced supernatant to a fresh microcentrifuge tube containing 1 µL of 500 mM DTT, centrifuge the sample, and vortex it before heating it for 5 min at 95 °C again.
7. Spin down the sample for 1 min at 12,000 x g; this is the reduced sample. Cool it down to room temperature and add 1.1 µL of 1 M NEM to both the reduced and the nonreduced sample. Vortex and spin down the samples.

3. SDS-PAGE

1. First, determine the appropriate SDS-PAGE resolving gel percentage for the protein of interest. For example, HIV-1 gp120, when deglycosylated, runs at ~60 kDa and is analyzed in a 7.5% gel.
2. Prepare the resolving gel mixture without TEMED according to the manufacturer's instructions (x% acrylamide, 375 mM Tris-HCl [pH 8.8,] 0.1% SDS [w/v], and 0.05% ammonium persulfate [APS] [w/v]) and degas under vacuum for >15 min. While the gel mixture is degassing, thoroughly clean the gel glass plates with 70% ethanol and lint-free tissues and place them into a casting apparatus.
3. Add TEMED to the resolving gel mixture (at a final concentration of 0.005% [v/v]), mix thoroughly, and pipette between glass plates, leaving ~1.5 cm space for the stacking gel. Carefully overlay the gel with deionized H₂O or isopropanol and leave it to polymerize.
4. Once the resolving gel has polymerized, prepare the stacking gel mixture (4% acrylamide, 125 mM Tris-HCl [pH 6.8], 0.1% SDS [w/v], and 0.025% APS [w/v]).
5. Flush the top of the resolving gel with deionized H₂O and, then, remove all water.
   NOTE: Use filter paper to remove the last drops.
6. Add TEMED to the stacking gel mixture (0.005% [v/v]), mix thoroughly, and overlay the resolving gel with stacking gel and insert a 15-well comb. Once the stacking gel has polymerized, transfer it to a running chamber and fill the upper and lower chambers with running buffer (25 mM Tris-HCl, 192 mM glycine [pH 8.3], and 0.1% SDS [w/v]).
7. Load 10 µL of sample per lane in a 15-lane minigel. Avoid loading the samples in the first and the last lane on the gel and load the nonreducing sample buffer in all empty lanes to prevent the smiling of bands. Run the gels at constant a 25 mA/gel until the dye front is at the bottom of the gel.
8. Remove the gels from the glass plates, stain the gels with protein-staining solution (10% acetic acid and 30% methanol in H₂O + 0.25% brilliant blue R250 [w/v]) for 5 min with agitation, and then, destain for 30 min with destaining solution (staining solution without brilliant blue R250).
9. Arrange the gels face-down on a plastic wrap and, then, place 0.4 mm chromatography paper on top of them. Place the gel sandwich chromatography paper-side down onto a gel dryer. Following the manufacturer's instructions, dry the gels for 2 h at 80 °C.
10. Transfer the dried gels to a cassette and overlay them with autoradiography film or phosphor screen. If using autoradiography film, this step must be performed in a dark room.

Results

The folding and secretion of HIV-1 gp120 from an adherent pulse chase is shown in Figure 2. The nonreducing gel (Cells NR in the figure) shows the oxidative folding of gp120. Immediately after the pulse labeling of 5 min (0 min chase) gp120 appears as a diffuse band higher in the gel, and as the chase progresses, the band migrates down the gel through even more diffused folding intermediates (IT) until it accumulates in the tight band (NT) that represents nat...

Discussion

Pulse-chase methods have been essential for developing scientists' understanding of protein folding in intact cells. While we have attempted to provide a method that is as general as possible, this approach has the potential for almost limitless variations to study various processes that occur during the folding, the transport, and the life of proteins inside the cell.

When performing a pulse chase using adherent cells in dishes, it is essential to treat each dish the same as much as possi...

Materials

Name	Company	Catalog Number	Comments
1.5 mL safeseal microcentrifuge tubes	Sarstedt	72.706.400
Acetic Acid	Sigma	A6283	glacial acetic acid
BAS Storage phosphor screen 20x25 cm	GE Life Sciences	28956475
Bromophenol Blue	Sigma	B8026	Molecular biology grade
Carestream Biomax MR films	Kodak	Z350370-50EA
Cell-culture media	Various	N/A	Normal cell culture media for specific cell-lines used
Cell-culture media, no methionine/cysteine	Various	N/A	Same media formulation as normal culture media e.g DMEM/MEM/RPMI, lacking methionine and cysteine
Charcoal filter paper	Whatman	1872047
Charcoal filtered pipette tips	Molecular bioproducts	5069B
Charcoal vacu-guard	Whatman	67221001
Coomassie Brilliant Blue R250	Sigma	112,553	for electrophoresis
Cysteine	Sigma	C7352	Molecular biology grade, Make 500 mM stock, store at -20
Dithiothreitol (DTT)	Sigma	10197777001	Molecular biology grade
EasyTag Express35S Protein Labeling Mix	Perkin Elmer	NEG772014MC	Other size batches of label are available depending on useage
EDTA	Sigma	E1644	Molecular biology grade
Gel-drying equipment	Various	N/A
Glycerol	Sigma	G5516	Molecular biology grade
Grade 3 chromatography paper	GE Life Sciences	3003-917
Hank's Balanced Salt Solution (HBSS)	Gibco	24020117
Kimwipes delicate task wipes	VWR	21905-026
MES	Sigma	M3671	Molecular biology grade
Methanol	Sigma	MX0490
Methionine	Sigma	M5308	Molecular biology grade, Make 250 mM stock, store at -20
Minigel casting/running equipment	Various	N/A
NaCl	Sigma	S7653	Molecular biology grade
N-ethylmaleimide	Sigma	E3876	Molecular biology grade, Make 1M stock in 100% ethanol, store at -20
PBS	Sigma	P5368	Molecular biology grade
Protein-A Sepharose fastflow beads	GE health-care	17-5280-04
Sodium Dodecyl Sulfate (SDS)	Sigma	L3771	Molecular biology grade
Triton X-100	Sigma	T8787	Molecular biology grade
Trizma base (Tris)	Sigma	T6066	Molecular biology grade
Typhoon IP Biomolecular imager	Amersham	29187194
Unwire Test Tube Rack 20 mm for waterbath	Nalgene	5970-0320PK