Cotranslational Translocation of Proteins into Microsomes Derived from the Rough Endoplasmic Reticulum of Mammalian Cells

I. INTRODUCTION
In mammalian cells, secretory proteins and membrane proteins of the organelles of the secretory pathway are initially transported across and integrated into the membrane of the rough endoplasmic reticulum (ER), respectively (for reviews, see Rapoport et al., 1996; Johnson and van Weas, 1999). This process can be studied in a cell-free translation/translocation system in which a protein of interest is synthesized and imported into microsomal membranes derived from the rough ER. Components used in such a system are rough microsomes (RM) usually prepared from dog pancreas, a cytosolic extract supporting protein synthesis, mRNA coding for a secretory or a membrane protein, and a radio-labeled amino acid, e.g., [³⁵S]methionine, for detection of the newly synthesized protein.

Upon translocation across the ER membrane, proteins become processed and modified, fold with the help of lumenal chaperones, and can assemble into oligomeric complexes (Lemberg and Martoglio, 2002; Daniels et al., 2003). These functions are maintained in rough microsomes and can be studied. This article describes the basic cell-free in vitro translocation system and provides methods to analyze the translocation of proteins across the microsomal membrane and integration into the lipid bilayer.

II. MATERIALS AND INSTRUMENTATION
Ambion: RNase inhibitor (Cat. No. 2684). Amersham Pharmacia: DEAE-Sepharose (Cat. No. 17-0709- 01), [³⁵S]methionine (Cat. No. AG 1594), and Sephacryl S-200 (Cat. No. 17-0584-10). Roche Applied Science: ATP (Cat. No. 1140965), CTP (Cat. No. 1140922), GTP (Cat. No. 1140957), UTP (Cat. No. 1140949), creatin kinase (Cat. No. 736988), creatin phosphate (Cat. No. 621714), and SP6 RNA polymerase (Cat. No. 810274). Fluka: EDTA (Cat. No. 03610), octaethylene glycol monododecyl ether (Cat. No. 74680), and SDS (Cat. No. 71725). Merck: Acetic acid (Cat. No. 100063), acetone (Cat. No. 100014), calcium chloride (Cat. No. 102389), HCl (Cat. No. 100317), isopropanol (Cat. No. 109634), magnesium acetate (Cat. No. 105819), magnesium chloride (Cat. No. 105833), 2-mercaptoethanol (Cat. No. 805740), potassium acetate (Cat. No. 104820), proteinase K (Cat. No. 124568), sodium acetate (Cat. No. 106268), sodium carbonate (Cat. No. 106392), sodium citrate (Cat. No. 106448), sucrose (Cat. No. 107654), and trichloroacetic acid (Cat. No. 100810). New England Biolabs: 7mG(5')ppp(5')G (Cat. No. 1404) and endoglycosidase H (Cat. No. 0702). Promega: Amino acid mixture minus methionine (Cat. No. L9961). Sigma: Dithiothreitol (DTT, Cat. No. D 0632), HEPES (Cat. No. H 3375), phenylmethylsulfonyl fluoride (PMSF, Cat. No. P 7626), Sephadex G-25 (Cat. No. G-25-150), Tris base (Cat. No. T 1503), and Triton X-100 (Cat. No. T 9284). Amersham Pharmacia: AKTAprime chromatography system (Cat. No. 18- 2237-18). Braun Biotech Int.: Potter S homogenizer. Qiagen: Plasmid Maxi Kit (Cat. No. 12162). Millipore: Amicon ultrafiltration unit 8050 (Cat. No. 5122) and YM100 ultrafiltration discs (Cat. No. 14422AM).

III. PROCEDURES
A. Preparation of Components Required for Cell-Free in vitro Protein Translocation across ER-Derived Rough Microsomes

1. Preparation of Rough Microsomes from Dog Pancreas
RMs can be prepared from most tissues or cells in culture. Dog pancreas, however, is the most convenient source for functional RMs because this tissue is specialized in secretion and contains an extended rough ER. Most importantly, pancreas from dog, in contrast to other animals, contains very little ribonuclease that would degrade the mRNA required for the translation reaction. Dogs may be obtained from an experimental surgery department or a pharmaceutical company. Ideally, the dogs are healthy and not operated or treated with drugs, and the pancreas is excised immediately after death.

Solutions
Glass and plasticware are autoclaved, and stock solutions and distilled water are either autoclaved or filtered through 0.45-µm-pore-sized filters.

1. Stock solutions:
   2 M sucrose: To make 500ml of the solution, dissolve 342.25g sucrose in water and complete to 500ml. Sucrose stock solution may be stored in aliquots at -20°C.
   
   1 M HEPES-KOH pH 7.6: To make 200 ml of the buffer, dissolve 47.7g HEPES in water, adjust pH to 7.6 with KOH solution, and complete to 200ml.
   
   4 M KOAc: To make 200 ml of the solution, dissolve 78.5 g KOAc in water, neutralize to pH 7 with diluted acetic acid, and complete to 100 ml.
   
   1 M Mg(OAc)₂: To make 100 ml of the solution, dissolve 21.5 g Mg(OAc)₂·4 H₂O in water, neutralize to pH 7 with diluted acetic acid, and complete to 100ml.
   
   500 mM EDTA: To make 100 ml of the solution, dissolve 14.7 g EDTA in water, adjust to pH 8.0 with NaOH solution and complete to 100 ml.
   
   1 M DTT: To make 10ml of the solution, dissolve 1.54 g DTT in water and complete to 10ml. Store in aliquots at -20°C.
   
   
   20mg/ml PMSF: To make 2ml of the solution, dissolve 40 mg PMSF in 2 ml isopropanol. Prepare just before use.


2. Homogenization buffer: 250mM sucrose, 50mM HEPES-KOH, pH 7.6, 50 mM KOAc, 6 mM Mg(OAc)₂, 1 mM EDTA, 1 mM DTT, and 30µg/ml PMSE To make 1 liter of the buffer, add 125ml 2M sucrose, 50ml 1M HEPES-KOH, pH 7.6, 12.5ml 4M KOAc, 6 ml 1M Mg(OAc)₂, 2 ml 500 mM EDTA, and complete to 1 liter with water. Add 1 ml 1M DTT and 1.5ml 20mg/ml PMSF to the cold buffer just before use.
3. Sucrose cushion: 1.3 M sucrose, 50 mM HEPES-KOH, pH 7.6, 50 mM KOAc, 6 mM Mg(OAc)₂, 1 mM EDTA, 1 mM DTT, and 10µg/ml PMSE To make 200ml of the solution, add 130ml 2M sucrose, 10ml 1M HEPES-KOH, pH 7.6, 2.5ml 4M KOAc, 1.2ml 1M Mg(OAc)₂, 0.4ml 500mM EDTA, and complete to 200ml with water. Add 0.2ml 1M DTT and 0.1ml 20mg/ml PMSF to the cold solution just before use.
4. RM buffer: 250 mM sucrose, 50 mM HEPES-KOH, pH 7.6, 50mM KOAc, 2mM Mg(OAc)₂, 1 mM DTT, and 10µg/ml PMSE To make 100ml of the buffer, add 12.5 ml 2M sucrose, 5 ml 1M HEPES-KOH, pH 7.6, 1.25 ml 4M KOAc, 0.2 ml 1M Mg(OAc)₂, and complete to 100ml with water. Add 0.1ml 1M DTT and 50µl 20mg/ml PMSF to the cold solution just before use.

Steps
This procedure is performed in the cold room. Samples and buffers should be kept on ice.

1. Excise the pancreas (10-15g) from a dog (e.g., beagle) and rinse in ~500ml ice-cold homogenization buffer.
2. Remove connective tissue and fat, and cut the pancreas into small pieces. (Optional: Shock-freeze the pieces in liquid nitrogen and store the tissue at -80°C.)
3. Place the pieces into ~120ml homogenization buffer and pass the tissue through a tissue press with a 1-mm mesh steel sieve.
4. Homogenize the pancreas in a 30-ml glass/Teflon potter with eight strokes at full speed (1500rpm).
5. Transfer homogenate into 30-ml polypropylene tubes. Centrifuge at 3000rpm (1000g) for 10min at 4°C in a Sorvall SS34 rotor.
6. Collect the supernatant, avoiding the floating lipid. Extract the pellet once more in 50 ml homogenization buffer as described in step 4 and centrifuge again (see step 5).
7. Transfer the two 1000-g supernatants to 30-ml polypropylene tubes. Centrifuge at 9500rpm (10,000g) for 10min at 4°C in a Sorvall SS34 rotor.
8. Collect the supernatant, avoiding the floating lipid and repeat step 7 (centrifugation at 10,000g) twice.
9. In the meantime, prepare four 70-ml polycarbonate tubes for a Ti45 rotor and add 25ml sucrose cushion per tube.
10. After the third 10,000g centrifugation (step 8), collect the supernatant and apply carefully, without mixing, onto the 25-ml sucrose cushions (see step 9).
11. Centrifuge at 35,000rpm (142,000g) for 1h at 4°C in a Beckman Ti45 rotor.
12. Discard the supernatant and resuspend the membrane pellet, the rough microsomes (RM), in 20ml RM buffer using a Dounce homogenizer.
13. Measure the absorption at 260 and 280nm of a 1: 1000 dilution of the RM suspension in 0.5% (w/v) SDS. Usually an absorption of 0.05-0.1 A₂₈₀/ml and a ratio A₂₆₀/A₂₈₀ of ~1.7 are obtained. When the pancreas is frozen (see step 2), the A₂₆₀/A₂₈₀ is 1.5-1.6.
14. Freeze 500-µl aliquots in liquid nitrogen and store at -80°C until use.

Note: RMs prepared by this procedure largely retain SRP on the membrane.

2. Preparation of Signal Recognition Particle (SRP)
Purification of components involved in protein translocation revealed that a cytosolic component, the signal recognition particle, is required for targeting nascent polypeptide chains to the ER membrane (Walter and Johnson, 1994). SRP is present in cytosolic extracts, but is also associated with RMs to a variable degree. In order to improve the efficiency of the translocation system, purified SRP may be added to the translation/translocation system. This is particularly useful when wheat germ extract is used for cellfree in vitro translation because this extract contains low amounts of functional SRP. SRP is isolated most conveniently from RMs by treatment with high salt. Released SRP is then purified by gel filtration and anion-exchange chromatography.

Solutions
Glass and plasticware are autoclaved, and stock solutions and distilled water are either autoclaved or filtered through 0.45-µm-pore-sized filters.

1. Stock solutions (see also preparation of RMs, Section III,A,1)
   
   1 M Tris-OAc, pH 7.5: To make 1 liter of the buffer, dissolve 121.1 g Tris base in water, adjust to pH 7.5 with acetic acid, and complete to 1 liter.
   
   
   10% (w/v) octaethylene glycol monododecyl ether: To make 10ml of the solution, dissolve 1 g of octaethylene glycol monododecyl ether in water and complete to 10ml.
2. RM buffer: As in solution 4 of Section III,A,1 (preparation of RMs).
3. High salt solution: 1.5M KOAc and 15mM Mg(OAc)₂. To make 50ml of the solution, add 18.75 ml 4M KOAc, 0.75ml 1M Mg(OAc)₂, and complete to 50ml with water.
4. Sucrose cushion: 500mM sucrose, 50mM Tris-OAc, pH 7.5, 500mM KOAc, 5mM Mg(OAc)₂, and 1 mM DTT. To make 100ml of the solution, add 25 ml 2M sucrose, 5 ml 1M Tris-OAc, pH 7.5, 12.5 ml 4M KOAc, 0.5ml 1M Mg(OAc)₂, and complete to 100ml with water. Add 0.1ml 1M DTT to the cold solution just before use.
5. Gel filtration buffer: 50mM Tris-OAc, pH 7.5, 250mM KOAc, 2.5mM Mg(OAc)₂, 1 mM DTT, and 0.01% octaethylene glycol monododecyl ether. To make 1 liter of the buffer, add 50ml 1M Tris-OAc, pH 7.5, 62.5 ml 4 M KOAc, 2.5 ml 1M Mg(OAc)₂, 1 ml 10% octaethylene glycol monododecyl ether, and complete to 1 liter with water. Add 1 ml 1M DTT to the cold solution just before use.
6. Washing buffer: 50mM Tris-OAc, pH 7.5, 350 mM KOAc, 3.5mM Mg(OAc)₂, 1 mM DTT, and 0.01% octaethylene glycol monododecyl ether. To make 100ml of the buffer, add 5ml 1M Tris-OAc, pH 7.5, 8.75 ml 4 M KOAc, 0.35 ml 1M Mg(OAc)₂, 0.1 ml 10% octaethylene glycol monododecyl ether, and complete to 100ml with water. Add 0.1 ml 1M DTT to the cold solution just before use.
7. SRP buffer: 50mM Tris-OAc, pH 7.5, 650mM KOAc, 6mM Mg(OAc)₂, 1 mM DTT, and 0.01% octaethylene glycol monododecyl ether. To make 100ml of the buffer, add 5ml 1M Tris-OAc, pH 7.5, 16.25ml 4M KOAc, 0.6ml 1M Mg(OAc)₂, 0.1ml 10% octaethylene glycol monododecyl ether, and complete to 100ml with water. Add 0.1ml 1M DTT to the cold solution just before use.

Steps
This procedure is performed in the cold room and samples and buffers should be kept on ice.

1. Prepare ER-derived rough microsomes from two dog pancreas as described previously and resuspend the final RM pellet (see step 8 in Section III,A,1) in 50ml RM buffer using a Dounce homogenizer. 2. Add 25ml high salt solution and incubate for 15 min at 4°C on a turning wheel.
2. Distribute the membrane suspension equally to two 70-ml polycarbonate tubes for a Ti45 rotor onto 25 ml sucrose cushion per tube, avoid mixing.
3. Centrifuge at 32,000rpm (120,000g) for 1 h at 4°C in a Beckman Ti45 rotor.
4. In the meantime, prepare ten 10.4-ml polycarbonate tubes for a Ti70.1 rotor and add 1ml sucrose cushion per tube.
5. After centrifugation (step 4), collect the supernatant and apply carefully, without mixing, onto the 1- ml sucrose cushions (see step 5).
6. Centrifuge at 65,000rpm (388,000g) for 1 h at 4°C in a Beckman Ti70.1 rotor.
7. Collect again the supernatant.
8. Concentrate the supernatant to approximately 10ml in a 50-ml Amicon ultrafiltration unit equiped with a YM100 membrane.
9. Load concentrated sample onto a Sephacryl S- 200 (2.6 × 20cm) equilibrated with gel filtration buffer and elute with gel filtration buffer. The flow rate is 1 ml/min. Follow elution with a UV monitor (1 = 280nm) by using, e.g., the ÄKTAprime chromatography system from Amersham Pharmacia.
10. Collect flow through (20-25 ml) and load immediately onto a DEAE-Sepharose column (1 × 3cm) equilibrated with gel filtration buffer. The flow rate is again 1 ml/min.
11. Wash column with 20 ml gel filtration buffer and 20ml washing buffer.
12. Elute SRP with SRP buffer and collect peak fraction (2-3 ml). The SRP eluate has an absorption of 1-4 A₂₆₀/ml and a ratio A₂₆₀/A₂₈₀ of approximately 1.4. Freeze 100-µl aliquots in liquid nitrogen and store at -80°C until use.

B. in vitro Translation and Translocation Assay
Solutions

1. Wheat germ extract: Wheat germ extract for cellfree in vitro translation is prepared as described by Erickson and Blobel (1983) except that we use a gel filtration buffer with lower potassium and magnesium concentrations [40mM HEPES-KOH, pH 7.6, 50mM KOAc, 1mM Mg(OAc)₂, 0.1% 2-mercaptoethanol]. Fresh wheat germ may be purchased from a local mill or from General Mills California. Store wheat germ in a desiccator over silica gel beads at 4°C. Considerable differences in translation efficiency may yield from different batches. Store wheat germ extract in 110-µl aliquots at -80°C and thaw only once.
2. Capped mRNA: To obtain mRNA coding for a secretory or a membrane protein, clone a cDNA encoding the protein of interest into a suitable expression vector downstream of a T7 or a SP6 promotor (e.g., pGEM from Promega Biotech). We generally prefer the SP6 promotor as the respective transcripts yield more efficient translation. Prepare plasmid DNA from Escherichia coli cultures using the Plasmid Maxi kit from Qiagen. For transcription, linearize the purified plasmid DNA with a suitable restriction enzyme that cuts downstream of the coding sequence. Alternatively, a template for transcription may be generated by polymerase chain reaction. Amplify the coding region of interest using Pfu DNA polymerase (Stratagene), an appropriate plasmid DNA as template, a forward primer containing the SP6 promotor, a Kozak sequence (Kozak, 1983) and a ATG for the initiation methionine, and a reverse primer starting with 5'- NNNNNNNNNCTA- to introduce a TAG stop codon at the desired position. Perform runoff in vitro transcription according to a standard protocol (e.g., in Sambrook et al., 1989) or by using a commercially available transcription kit (e.g., mMESSAGAmMACHINE kits from Ambion Cat. No. 1340 and 1344). Dissolve the resulting capped messenger RNA in water after extraction with phenol and chloroform and precipitation with sodium acetate and ethanol. Store mRNA at -80°C.
3. Energy mix: 50mM HEPES-KOH, pH 7.6, 12.5mM ATP, 0.25mM GTP, 110mM creatine phosphate, 10mg/ml creatine kinase, and 0.25 mM of each amino acid except methionine. To make 1 ml of the solution, dissolve 41 mg creatin phosphate (disodium salt · 4H₂O) and 10mg creatin kinase in 590µl water and add 50µl 1M HEPES-KOH, pH 7.6, 125 µl 100mM ATP solution (Roche Applied Science), 2.5µl 100mM GTP solution (Roche Applied Science), and 250µl 19 amino acids mix without methionine (Promega, 1 mM of each amino acid). Store the energy mix in 22µl aliquots at -80°C. Do not refreeze!
4. Salt mix: 500mM HEPES-KOH, pH 7.6, 1M KOAc, and 50 mM Mg(OAc)₂. To make 1 ml of the solution, mix 500µl 1M HEPES-KOH, pH 7.6, 250µl 4M KOAc, 70µl 1M Mg(OAc)₂, and 180µl water.
5. SRP buffer: As in solution 7 of Section III,A,2. (preparation of SRP).
6. 20% (^w/_v) trichloroacetic acid: To make 100ml of the solution, dissolve 20 g trichloroacetic acid in water and complete to 100 ml.

Steps

1. Per assay (25 µl), mix on ice 6 µl water, 1 µl salt mix, 2 µl energy mix, 10µl wheat germ extract, 2µl SRP buffer or SRP solution (see Section III,A,2, step 12), 2µl RM suspension (see Section III,A,1, step 8), 1 µl [³⁵S]methionine (>1000Ci/mmol), and 1µl capped mRNA.
2. Incubate for 60min at 25°C.
3. Add 25µl (1 volume) 20% trichloroacetic acid to precipitate proteins and centrifuge at 14,000rpm for 3min at room temperature in an Eppendorf centrifuge.
4. Discard supernatant. Wash pellet with 150µl cold acetone, and centrifuge as in step 3.
5. Discard supernatant and repeat step 4.
6. Discard supernatant, centrifuge tube shortly, and remove residual acetone completely with a pipette.
7. Add 20-30 µl sample buffer for SDS-polyacrylamide gel electrophoresis and heat for 10 min at 65°C.
8. Load 5-10µl onto a SDS-polyacrylamide gel and analyze the sample by electrophoresis. Radiolabelled proteins can be visualized by autoradiography, fluorography, or on a phosphorimager.

Controls: Translation without mRNA (add water instead); translation without membranes (add RM buffer instead, see solution 4 in Section III,A,1).

Note: Translation and translocation assays have to be optimized for each mRNA with respect to magnesium and potassium concentrations as well as to the amount of mRNA, SRP, and membranes. Optimal salt concentrations vary from 1 to 3.5 mM magnesium and from 70 to 150mM potassium and are adjusted by using adapted salt mixes. The amount of SRP and membranes may be varied by diluting SRP and RM solutions with the respective buffers.

C. Assays to Characterize the Translocation Products
1. Protease Protection Assay
Proteinase K is used to test the translocation of proteins or parts of proteins across microsomal membranes (Blobel and Dobberstein, 1975). Membranes are impermeable to the protease and therefore only proteins or protein domains exposed on the cytoplasmic side of the microsomes are digested. To demonstrate that only intact microsomal vesicles protect lumenal proteins or protein domains, nonionic detergent (e.g., Triton X-100) is added to open the membrane.

Protease treatment is also used to characterize the topology of membrane proteins. In this case, protease treatment is often followed by immunoprecipitations with antibodies directed against defined regions of the protein investigated. Successful immunoprecipitations indicate that the respective domains are exposed on the lumenal side of the microsomes and are protected from the protease.

Solutions

1. Sucrose cushion: 500mM sucrose, 50mM HEPESKOH, pH 7.6, 50mM KOAc, 2mM Mg(OAc)₂, and 1 mM DTT. To make 10ml of the buffer, add 1.25ml 2M sucrose, 0.5ml 1M HEPES-KOH, pH 7.6, 125µl 4M KOAc, 20 µl 1M Mg(OAc)2, 10 µl 1M DTT, and complete to 10ml with water.
2. 3 mg/ml proteinase K: To make 1 ml of the solution, dissolve 3 mg proteinase K in 1 ml water.
3. 20mg/ml PMSF solution: As is solution 1 of Section III,A,1 (preparation of RMs).
4. 10% (^w/_v) Triton X-100: To make 10ml of the solution, dissolve 10 g Triton X-100 in water and complete to 10ml.
5. 20% (^w/_v) trichloroacetic acid: As in solution 6 of Section III,B.

Steps

1. Perform a translation/translocation assay (25µl) as described in Section III,B (steps 1 and 2).
2. In the meantime, prepare a 200-µl thick-wall polycarbonate tube for a TLA100 rotor and add 100-µl sucrose cushion.
3. After translation/translocation, apply the reaction mixture carefully onto the 100-µl sucrose cushion.
4. Wash microsomes by centrifugation through the sucrose cushion at 48,000 rpm (~100,000 g) for 3 min at 4°C in a Beckman TLA100 rotor.
5. Remove the supernatant with a pipette.
6. Resuspend the microsome pellet in 25µl RM buffer (see solution 4 in Section III,A,1) and split the sample in three 8-µl aliquots.
7. 1. To the first aliquot (mock treatment) add 2µl water.
   2. To the second aliquot add 1 µl proteinase K solution (3mg/ml) and 1 µl water.
   3. To the third aliquot add 1 µl proteinase K solution (3mg/ml) and 1 µl 10% Triton X-100.
8. Incubate the samples for 10min at 25°C.
9. Stop proteolysis by adding 1 µl 10mg/ml PMSF per sample.
10. Add 40 µl water and 50 µl20% trichloroacetic acid to each sample to precipitate protein.
11. Centrifuge samples, wash protein pellets with acetone, and analyze samples by SDS-polyacrylamide gel electrophoresis as described in Section III,B, steps 3-8.

2. Sodium Carbonate Extraction
By alkaline treatment with sodium carbonate at pH 11, microsomal membranes are opened and release their content and peripherally associated proteins. The method is used to separate these proteins from proteins integrated into the lipid bilayer (Fujiki et al., 1982).

Solutions

1. 0.1 M Na₂CO₃: To make 10 ml of the solution, dissolve 106 mg Na₂CO₃ in water and complete to 10ml. Prepare just prior to use.
2. Alkaline sucrose cushion: 0.1M Na₂CO₃ and 250mM sucrose. To make 10ml of the solution, dissolve 106 mg Na2CO3 in water, add 1.25 ml 2M sucrose (see solutions 1 of Section III,A,1), and complete to 10 ml with water.
3. 20% (^w/_v) trichloroacetic acid: As in solution 6 of Section III, B.

Steps

1. Perform a translation/translocation assay (25µl) as described in Section III,B (steps 1 and 2).
2. Wash microsomes by centrifugation through a sucrose cushion as described in Section III,C,1, steps 2-5.
3. Resuspend microsome pellet in 25µl carbonate solution and incubate for 15 min on ice.
4. In the meantime, prepare a 200-µl thick-wall polycarbonate tube for a TLA 100 rotor and add 100-µl alkaline sucrose cushion.
5. For centrifugation, apply the sample (step 3) carefully onto a sucrose cushion in the polycarbonate tube.
6. Centrifuge at 55,000rpm (130,000g) for 10min at 4°C in a Beckman TLA 100 rotor.
7. Recover the supernatant and pellet.
8. 1. To the supernatant add 150µl 20% trichloroacetic acid to precipitate proteins. Centrifuge the sample, wash the protein pellet with acetone, and prepare the sample for SDS-polyacrylamide gel electrophoresis as described in Section III, B, steps 3-8.
   2. To the pellet add directly 20-30µl sample buffer for SDS-polyacrylamide gel electrophoresis (see Section III,B, steps 7 and 8).
9. Analyze samples by SDS-polyacrylamide gel electrophoresis and autoradiography or phosphorimaging.

3. Inhibition of N-Glycosylation with Glycosylation Acceptor Tripeptide
The recognition sites for N-glycosylation in the ER are Asn-X-Ser and Asn-X-Thr. In the cell-free in vitro translation/translocation system described herein, the tripeptide N-benzoyl-Asn-Leu-Thr-methylamide efficiently competes with newly synthesized proteins for N-glycosylation (Lau et al., 1983). The translocated protein is therefore not glycosylated in the presence of acceptor tripeptide, and the effects of oligosaccharides, e.g., on protein folding and assembly, may be investigated.

Solution
Acceptor tripeptide solution: Synthesize N-Benzoyl- Asn-Leu-Thr-methylamide on a peptide synthesizer. Dissolve the tripeptide in methanol at a concentration of 0.5 mM (0.23 mg/ml) and store the stock solution at -80°C.

Steps

1. Evaporate per translation/translocation assay (25µl) 1.5µl acceptor tripeptide solution in a test tube using a Speed-Vac centrifuge.
2. Add the components for the translation/translocation assay to the tripeptide, vortex gently, and perform the assay as described in Section III,B (steps 1 and 2).
3. Precipitate the sample with trichloroacetic acid and analyze by SDS-polyacrylamide gel electrophoresis and autoradiography (see Section III,B, steps 3-8).

4. Endoglycosidase H Treatment
Treatment with endoglycosidase H is used to test N-glycosylation of proteins translocated into microsomal membranes. The glycosidase cleaves oligosaccharides of the high mannose type from glycoproteins, leaving an N-acetylglucosamine residue attached to the polypeptide (Tarentino et al., 1974).

Solutions

1. Denaturing solution: 0.5% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. To make 10ml of the solution, dissolve 50mg SDS in water, add 100µl 2- mercaptoethanol, and complete to 10 ml with water.
2. 0.5 M Na-citrate, pH 5.5: To make 10 ml of the buffer, dissolve 1.47 g Na₃-citrate. 2 H₂O in water, adjust to pH 5.5 with HCl solution, and complete to 10ml.

Steps

1. Perform a translation/translocation assay (25µl) as described in Section III,B (steps 1 and 2).
2. Wash microsomes by centrifugation through a sucrose cushion as described in Section III,C,1, steps 2-5.
3. Resuspend microsome pellet in 25µl denaturing solution and incubate for 10min at 95°C.
4. Add 2.8 µl reaction buffer and 1 µl endoglycosidase H (1000U/µl). Incubate for 1h at 37°C.
5. Precipitate the sample with trichloroacetic acid and analyze by SDS-polyacrylamide gel electrophoresis and autoradiography or phorphorimaging (see Section III,B, steps 3-8).

IV. COMMENTS
As an alternative to wheat germ extract, commercially available rabbit reticulocyte lysate (e.g., from Promega) may be used for cell-free in vitro translation. Reticulocyte lysate contains sufficient SRP and therefore no additional SRP is usually required for optimization of translocation. When reticulocyte lysate is used, however, RMs should be treated with microccocal nuclease to digest endogenous mRNA (Garoff et al., 1978). The reticulocyte translation machinery will otherwise promote completion of nascent pancreatic secretory proteins.

References
Blobel, G., and Dobberstein, B. (1975). Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67, 852-862.

Daniels, R., Kurowski, B., Johnson, A. E., and Hebert, D. N. (2003). N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin. Mol. Cell 11, 79-90.

Erickson, A. H., and Blobel, G. (1983). Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 96, 38- 50.

Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, R B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum. J. Cell Biol. 93, 97-102.

Garoff, H., Simons, K., and Dobberstein, B. (1978). Assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro. J. Mol. Biol. 124, 587-600.

Johnson, A. E., and van Waes, M. A. (1999). The translocon: A dynamic gateway at the ER membrane. Annu. Rev. Cell Dev. Biol. 15, 799-842.

Kozak, M. (1983). Comparison of initiation of protein synthesis in procaryotes, eukaryotes and organelles. Microbiol. Rev. 47, 1-45.

Lau, J. T. Y., Welply, J. K., Shenbagamurthi, P., Naider, E, and Lennarz, W. J. (1983). Substrate recognition by oligosaccharyl transferase: Inhibition of translational glycosylation by acceptor peptides. J. Biol. Chem. 258, 15255-15260.

Lemberg, M. K., and Martoglio, B. (2002). Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis. Mol. Cell 10, 735-744.

Rapoport, T. A., Jungnickel, B., and Kutay, U. (1996). Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu. Rev. Biochem. 65, 271-303.

Sambrook, J., Fritsch, E. E, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY.

Tarentino, A. L., Trimble, R. B., and Maley, E (1978). Endo-β-Nacetylglucosaminidase from Streptomyces plicatus. Methods Enzymol. 50, 574-580.

Walter, P., and Johnson, A. E. (1994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 10, 87-119.