Molecular Techniques and Methods

Isolation of Mitochondria for in Organello Protein Synthesis

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

When isolating mitochondria for protein synthesis, the emphasis is placed on preparing intact, coupled mitochondria rather than on the yield.

In this method, mitochondria is separated by differential centrifugation from the bulk of nuclei, plastids, and cellular debris which differ in particle size or density. Mitochondria are then further separated from the remaining plastids and nuclear debris by gradient centrifugation. Homogenization media for mitochondrial isolation contain an osmoticum, EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone (PVP) which binds phenolics.

  • Mitochondrial yield: 50 ug of protein/ gram of packed suspension cells





  • MATERIALS AND SOLUTIONS

    Cell Grinding Buffer (1 liter)
    0.4 M mannitol ------------------------------------ 72.9 g
    50 mM Tricine-NaOH (pH 7.8) ------------------- 50 ml of 1 M Tricine-NaOH
    1 mM EGTA --------------------------------------- 2 ml of 0.5 M EGTA
    0.1% BSA (w/v) ----------------------------------- 1 g
    20 mM 2-mercaptoethanol ------------------------- 1.4 ml of 14.4 M 2-mercaptoethanol
    Add deionized H2O to make a final volume of ----- 1 liter


    Rinse Buffer (1 liter)
    0.4 M mannitol -------------------------------------- 72.9 g
    10 mM Tricine-NaOH (pH 7.5) -------------------- 10 ml of 1 M Tricine-NaOH
    1 mM EGTA --------------------------------------- 2 ml of 0.5 M EGTA
    0.1% BSA ------------------------------------------ 1 g
    Add deionized H2O to make a final volume of ----- 1 liter


    1.6 M Sucrose Step Gradient Buffer (100 ml)
    1.6 M Sucrose ------------------------------------- 54.8 g
    10 mM Tricine-NaOH (pH 7.2) -------------------- 1 ml of 1 M Tricine-NaOH
    1 mM EDTA --------------------------------------- 0.2 ml of 0.5 M EDTA
    0.1% BSA ------------------------------------------ 0.1 g
    Add deionized H2O to make a final volume of ----- 100 ml



    1.2 M Sucrose Step Gradient Buffer (100 ml)
    1.2 M Sucrose ------------------------------------- 41.4 g
    10 mM Tricine-NaOH (pH 7.2) -------------------- 1 ml of 1 M Tricine-NaOH
    1 mM EDTA --------------------------------------- 0.2 ml of 0.5 M EDTA
    0.1% BSA ------------------------------------------ 0.1 g
    Add deionized H2O to make a final volume of ----- 100 ml


    0.6 M Sucrose Step Gradient Buffer (100 ml)
    0.6 M Sucrose -------------------------------------- 20.6 g
    10 mM Tricine-NaOH (pH 7.2) -------------------- 1 ml of 1 M Tricine-NaOH
    1 mM EDTA --------------------------------------- 0.2 ml of 0.5 M EDTA
    0.1% BSA ------------------------------------------ 0.1 g
    Add deionized H2O to make a final volume of ----- 100 ml


    Dilution Buffer (100 ml)
    0.3 M mannitol ----------------------------------- 18.2 g
    10 mM Tricine-NaOH (pH 7.2) ------------------ 1 ml of 1 M Tricine-NaOH
    1 mM EGTA ------------------------------------- 0.2 ml of 0.5 M EGTA
    Deionized H2O to make a final volume of ------- 100 ml


    MET (100 ml)
    0.4 M Mannitol ---------------------------------- 72.9 g
    1 mM EDTA ------------------------------------- 0.2 ml of 0.5 M EDTA
    10 mM Tricine-NaOH (pH 7.2) ------------------ 1 ml of 1 M Tricine-NaOH
    Deionized H2O to make a final volume of ------- 100 ml
  • Keep at 4oC.




    PROCEDURES

  • All steps should be performed in ice!

    1. Collect 100-200 g cells from rapidly growing cultures by centrifugation for 5 minutes at 1,500g and 4oC.

    2. Resuspend the cells in 1 volume of Cell Grinding Buffer.

    3. Break suspension cultures in a Bead-Beater with ice in outer jacket. Three times for 10 second-pulse at high speed.

    4. Filter the homogenate through four layers of cheesecloth and one layer of Miracloth.

    5. Centrifuge the homogenate for 5 minutes at 1,500g and 4oC to remove cell debris, plastids, and nuclei.

    6. Transfer the supernatant to a new centrifuge tube.

    7. Centrifuge for 15 minutes at 15,000g and 4oC.

    8. Gently resuspend the crude mitochondrial pellet in the small volume of supernatant that remains after decanting.

    9. Next wash the mitochondria by adding 5-10 ml of Rinse Buffer.

    10. Homogenize the pooled suspension several times with a loose-fitting homogenizer.

    11. Centrifuge the homogenate for 5 minutes at 1,200g and 4oC.

    12. Transfer the supernatant to a new tube.

    13. Centrifuge for 15 minutes at 15,000g and 4oC.

    14. The resulting pellet will be soft, so remove the supernatant with a pipette.

    15. Resuspend the pellet in the remaining supernatant with a brush.

    16. Dilute the suspension with Rinse Buffer, and use a loose-fitting homogenizer to make a uniform suspension.

    17. Layer 3-4 ml of mitochondrial suspension per sucrose step gradient consisting of 10 ml 1.6 M Sucrose Step Gradient Buffer, 10 ml 1.2 M Sucrose Step Gradient Buffer, and 10 ml 0.6 M Sucrose Step Gradient Buffer.

    18. Centrifuge the gradients at 25,000 rpm for 1 hour.

    19. Collect the lower 1.2 M
    ||1.6 M interface band.
  • The upper 0.6 M||1.2 M interface band tends to be more contaminated with plastid DNA. However, the upper band does exhibit protein synthesis activity.

    20. Dilute the mitochondrial fraction with 3 volumes of Dilution Buffer slowly over a 15-minute period to minimize disruption by osmotic shock.

    21. Collect the mitochondria by centrifugation for 15 minutes at 15,000g and 4oC.

    22. Discard the supernatant and drain the tubes well.

    23. Gently resuspend the pellet in 1 ml MET.

    24. Determine the protein concentration and dilute the mitochondria to a concentration of 5-10 ug protein/ ul.
  • The mitochondria are then ready to be assayed for protein synthesis activity.




    NOTES

  • Contamination of mitochondrial preparation by other cellular fractions capable of in vitro protein synthesis can be monitored by including selective protein synthesis inhibitors in incubation solutions. Cycloheximide (5 ug/ml) inhibits the activity of cytoplasmic 80 S ribosomes, while erythromycin has been reported to inhibit plastid protein synthesis but not plant mitochondrial protein synthesis.





  • KIT INFORMATION




    REFERENCES

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  • Hack, E, Leaver, CJ (1984) Curr. Genet. 8: 537.

  • Ku, HS, Pratt, HK, Spuff, AR, Harris, WM (1968) Plant Physiol. 43, 883.


  • Murray, MG, Thompson, WF (1980) Nucleic Acids Res. 8: 4321.


  • Palmer, D, Shields, CR (1984) Nature (London) 307: 437.

  • Powling, A (1981) Mol. Gen. Genet. 193: 82.


  • Qudtier, F, Vedel, F (1977) Nature (London) 268: 365.


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  • Vedel, F, Mathieu, C (1982) Anal. Biochem. 127: 1.



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