Cotranslational Translocation of Proteins into Canine Rough Microsomes

Harris D. Bernstein1

1 National Institute of Diabetes and Digestive and Kidney Disesases/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.4
DOI:  10.1002/0471143030.cb1104s00
Online Posting Date:  May, 2001
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Abstract

Rough microsomes (RMs) are small vesicular fragments of the rough endoplasmic reticulum (ER). They can be used in cell‐free protein translocation studies to analyze ER targeting signals, signal sequence cleavage, N‐linked glycosylation, and membrane protein biogenesis. This unit describes preparation and use of RMs.Rough microsomes (RMs) are small vesicular fragments of the rough endoplasmic reticulum (ER). They can be used in cell‐free protein translocation studies to analyze ER targeting signals, signal sequence cleavage, N‐linked glycosylation, and membrane protein biogenesis. This unit describes preparation and use of RMs.

     
 
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Table of Contents

  • Basic Protocol 1: Translocation into Canine Rough Microsomes
  • Support Protocol 1: Preparation of RMs from Canine Pancreas
  • Support Protocol 2: Preparation of EDTA‐Stripped Rough Microsomes
  • Support Protocol 3: Preparation of Column‐Washed Rough Microsomes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Translocation into Canine Rough Microsomes

  Materials
  • Wheat germ or reticulocyte translation mix (see unit 11.2)
  • 1 eq/µl RM solution (see protocol 2)
  • mRNA encoding a protein of interest
  • mRNA encoding a control cytoplasmic protein
  • Buffer C (see recipe)
  • 30% trichloroacetic acid (TCA; store at 4°C), ice cold
  • For optional steps:
  • 20 mg/ml proteinase K (store at −20°C)
  • Nonionic detergent: e.g., 20% Triton X‐100 or Nonidet P‐40 (NP‐40; store at −20°C)
  • 25% sodium dodecyl sulfate (SDS; store at room temperature)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • Buffer D (see recipe)
  • Endoglycosidase H (purchased as solid or stock solution)
  • 100 mM sodium carbonate, pH 11.5, ice cold
  • Glacial acetic acid
  • 1× SDS sample buffer ( appendix 2A)
  • Water bath or heating block, 26° or 30°C and (for optional step) 100°C
  • Tabletop ultracentrifuge and rotor (e.g., Beckman TLA 100.2 rotor; for optional step)
  • Additional reagents and solutions for in vitro translation (unit 11.2) and one‐dimensional SDS‐PAGE (unit 6.1)

Support Protocol 1: Preparation of RMs from Canine Pancreas

  Materials
  • Buffer A (see recipe), ice cold
  • Dog (e.g., beagle weighing 10 to 12 kg)
  • Buffer B (see recipe), ice cold
  • Buffer C (see recipe), ice cold
  • 1% (w/v) SDS ( appendix 2A), ice cold
  • Liquid nitrogen
  • Dissecting tools: small scissors, regular and fine forceps, scalpel (with new blade)
  • Small plastic cutting board
  • Single‐edge razor blades (at least 5)
  • Potter‐Elvejhem‐style tissue homogenizer (e.g., B. Braun Biotech) with 60‐ml cylinder and matched Teflon plunger
  • 50‐ml polycarbonate or polypropylene centrifuge tubes (∼28.5‐mm diameter)
  • Medium‐speed centrifuge and rotor (Sorvall SS‐34 or Beckman JA20, or equivalent)
  • 25 × 89–mm (26‐ml) polycarbonate ultracentrifuge bottles with tops
  • 10‐ml glass syringe fitted with a round‐tipped pipetting needle
  • Ultracentrifuge and rotor (Beckman Ti 50.2 or equivalent)
  • 60‐ml Dounce homogenizer fitted with type A pestle

Support Protocol 2: Preparation of EDTA‐Stripped Rough Microsomes

  • Crude RM fraction (see protocol 2)
  • Buffer C (see recipe)/50 mM EDTA, ice cold
  • recipeBuffer C/0.5 M sucrose, ice cold

Support Protocol 3: Preparation of Column‐Washed Rough Microsomes

  • Sepharose CL‐2B resin (Pharmacia Biotech)
  • Buffer E (see recipe), ice cold
  • Crude RM fraction (see protocol 2)
  • Chromatography column of volume equivalent to ten times the volume of RMs to be treated
  • Ultracentrifuge and rotor (Beckman 45 Ti rotor or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Blobel and Dobberstein, 1975. See above.
  Landmark paper containing the first clear demonstration that a presecretory protein synthesized in a cell‐free translation system is transported vectorially into ER vesicles.
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