Topic 11.1

Isolation of Mitochondria

Ian M. Møller, Royal Veterinary and Agricultural University, Denmark; Allan G. Rasmusson, Lund University, Sweden

Although it is possible to measure respiration by intact tissues it is often useful to be able to isolate uncontaminated, intact and functional mitochondria. Here, we will consider some of the principles for isolating cell organelles. This discussion is not meant to be exhaustive nor will it be a cookbook with recipes to be followed step-by-step. Useful general references are Douce 1985, Møller et al. 1996, and a number of chapters in Volume 148 of Methods in Enzymology from 1987.

Choice of Material

If the aim of the experiment is to investigate a specific physiological problem, then the plant material is often well-defined. However, in many cases there is some flexibility in the choice of material and it is therefore wise to follow certain rules of thumb:

Choose a tissue that is easy to obtain in relatively large amounts (at least 100–200 g fresh weight), such as tubers or hypocotyls.
Choose a tissue for which a good protocol is available. It can take quite a lot of time and effort to optimize an isolation protocol.

Note that the isolation of mitochondria from green tissues presents special problems because of the large amount of chloroplasts. Roots are a notoriously difficult tissue for isolation of mitochondria.

Disruption of the Tissue

The aim is to rupture as many of the cells in the tissue as possible in order to release their cytoplasm with the mitochondria. Whereas many mammalian tissues are easy to homogenize, plant tissues are much tougher mainly because of the cell walls. There are a number of standard homogenizers, including mixers with rotating knives or razor blades, juice extractors, ball mills, and mortar and pestle; the most suitable method for the specific plant material should be chosen. This may involve some initial optimization experiments.

Homogenization Medium

The medium for homogenizing rat liver only contains an osmoticum and a buffer. That will not work with plant tissues because of the disruption of the large central vacuole (absent in mammalian cells), which releases its fairly acidic content often containing phenols and other potentially harmful substances.

Let us consider the components of the optimal homogenization medium for isolating intact and functional organelles from plant material, one by one.

Osmoticum. This is required to prevent the mitochondria from losing their matrix content because of swelling and bursting. For the isolation of plant mitochondria, there is a tradition of using sucrose or mannitol at 0.2–0.5 M; whereas 0.15 M KCl is standard for the isolation of mammalian mitochondria. In neither case does this mimic precisely the conditions in the cytosol, but it works.

Buffer. The pH in the cytosol of plant cells is around 7.5, and the homogenate should be buffered at pH 7–8. A buffer should therefore be included and a plethora of different buffers are available. Choose an inexpensive one with a pKa of 7–8 (e.g., 4-morpholinopropane sulfonic acid = MOPS) and be sure to use enough to counterbalance the acidic vacuolar content. The tissue type, the tissue/medium ratio, and the buffer concentration determine whether the acidic vacuolar content is properly neutralized. Obviously, special tissues like lemons or CAM plants harvested late at night or in the early morning, require special treatment!

Metal ion chelators. EDTA is usually included to bind mainly Ca²⁺ and Mg²⁺, which may otherwise activate proteolytic enzymes.

Antioxidants. Antioxidants (reductants) such as ascorbic acid, dithiothreitol, or cysteine are used mainly to protect sulfhydryl groups on proteins against oxidation.

Bovine serum albumin (BSA). This protein from the blood of cows is usually included to protect the mitochondria: BSA binds fatty acids (its function in the blood) released by the action of phospholipases on phospholipids. BSA may also bind phenolics that are released from the vacuole. Both of these groups of compounds can otherwise be troublesome later. In addition, BSA may prevent proteases from degrading mitochondrial proteins by serving as an alternative substrate present at high concentrations.

Polyvinylpyrrolidone (PVP). A polymer that is often included, especially when using leaf material, to adsorb phenolic compounds.

Filtration

To remove larger tissue fragments, cell walls, and often starch grains, the homogenate is always filtered, usually through a nylon net with a mesh size of 30–100 µm.

Purification of Mitochondria

The first step of the purification is usually differential centrifugation. This means that the filtered homogenate is centrifuged first at low speed for a short time (e.g., 3000 g for 5–10 min.) and the pellet (containing cell wall fragments, starch grains, nuclei, intact plastids) is discarded. The supernatant is then centrifuged faster and for a longer time (e.g., 10,000 g for 10–20 min.) and the pellet (termed crude mitochondria) is collected. It contains intact and damaged mitochondria as well as peroxisomes and plastid membranes. The supernatant is discarded.

As soon as the mitochondria have been pelleted and thus removed from potentially damaging soluble substances in the homogenate, the medium can be greatly simplified. Usually the so-called wash medium contains only osmoticum and weak buffer (no need for a strong buffer since there is no vacuole content to neutralize). However, it may be wise to include EDTA to bind divalent cations as well as BSA to adsorb any fatty acids released by the action of phospholipases. The resuspended crude mitochondria are often diluted to 50–100 ml with wash medium and subjected to another round of slow and fast centrifugation to remove more contaminants.

The aim of the next step is to separate the intact mitochondria from the contaminants. This usually involves a density gradient centrifugation with Percoll either alone (e.g., Struglics et al. 1993) or together with other dense materials (e.g., Day et al. 1985). Percoll (colloidal silica coated with PVP) is inert and osmotically inactive. It has the further advantage that a Percoll gradient is self-generating because the Percoll microspheres pellet very slowly during the centrifugation. The crude mitochondria are layered on top of the wash medium containing the appropriate concentration of Percoll (usually 25–30%). If the Percoll concentration and the speed and duration of the centrifugation have been chosen correctly, the intact mitochondria will form a distinct band, well away from the contaminants. This band can be collected, washed free of Percoll by repeated dilution and centrifugation, and either used immediately, or frozen with 5% (v/v) dimethylsulfoxide (to preserve integrity) in liquid nitrogen and stored at -80°C until use. This preparation can be stable for months, retaining the ability to form a proton electrochemical gradient over the inner membrane.

The yield is 1–50 mg mitochondrial protein per 100 g fresh weight, depending on the tissue. This represents only a small fraction of the total amount of mitochondria in the tissue.

The purification of mitochondria from green leaves presents special problems and a good protocol is described in Day et al. (1985). There is, at present, no good protocol for the isolation of both pure and functional mitochondria from Arabidopsis leaves (Hausmann et al. 2003; Keech et al. 2005), but a protocol for the purification of mitochondria from Arabidopsis cell cultures is given in Kruft et al. (2001). Finally, Boutry et al. (1984) demonstrate how purified mitochondria can be isolated from small amounts of plant tissue (down to 0.5 g fresh weight).

Subfractionation of Mitochondria

The mitochondrion contains four compartments, each with a unique complement of proteins: the outer membrane (containing around 7% of the protein), the intermembrane space (perhaps 1–2%), the inner membrane (50–60%) and the matrix (30–40%). It is sometimes desirable to be able to isolate these, for instance when trying to localize a given function within the mitochondrion and is usually done in the following way (e.g., Agius et al. 2001) (Figure 1):

Figure 1 Subfractionation of mitochondrion compartments. The hypotonic medium causes the mitochondria to swell because the matrix takes up water. Since the surface area of the inner membrane (IMM) greatly exceeds that of the outer membrane (OMM), the latter breaks, releasing the contents of the intermembrane space (IMS) together with some OMM. Addition of osmoticum causes the mitoplasts—mitochondria minus (some) OMM—to contract, making them less susceptible to mechanical damage. The mitoplasts can be pelleted and subfractionated into IMM vesicles (submitochondrial particles or SMP) and matrix by sonication followed by differential centrifugation. When sonication is done in a high-salt medium, the SMP are inside-out—that is, they have the opposite orientation to that of the IMM in intact mitchondria (Møller et al. 1987). The SMP are contaminated with some OMM. The OMM are isolated from the mitoplast supernatant by centrifugation. The yield of OMM is small, but relatively uncontaminated by IMM. Intermembrane proteins are found in the OMM supernatant. Symbols: Small squares—matrix protein (e.g., malate dehydrogenase), triangles—intermembrane space protein (no known marker for plant mitochondria), square-and-circle—F_oF₁-ATPase (ATP synthase) in the inner membrane with the F₁ facing the matrix. (Click image to enlarge.)

The outer membrane is ruptured, either by osmotic swelling or by selective solubilization with digitonin, a special detergent. In both cases, it is critical to avoid rupturing the inner membrane because the release of matrix from even a few percent of the mitochondria will cause very large contamination of the intermembrane space, which is small in volume.
Pellet the mitochondria. The supernatant contains intermembrane space plus outer membrane from at least a fraction of the mitochondria (usually only 10–20%). The outer membrane can be pelleted from this supernatant and the intermembrane space proteins are left in solution.
The pelleted mitochondria—often called mitoplasts, which are mitochondria without outer membrane—are then disrupted, (e.g., by sonication), and the inner membranes pelleted. That leaves the matrix proteins in the supernatant. The inner membrane usually reseals after rupture and forms sealed vesicles, (so-called submitochondrial particles) the sidedness of which can vary depending on the disruption method and conditions (Møller et al. 1987). Note that the inner membrane fraction usually contains some outer membrane contamination (e.g., Agius et al. 2001).

Methods for Establishing Purity, Intactness, and Functionality of the Fractions

It is often important to determine the degree of purity of the mitochondria and their subfractions (Agius et al. 2001).

The intactness of the mitochondria is usually established with enzyme assays that take advantage of the fact that an intact membrane is not permeable to certain molecules. For instance, the outer mitochondrial membrane is impermeable to cytochrome c, a 12.5 kDa protein that is too large to pass through the outer membrane pores, and NADH cannot pass through an intact inner membrane.

Functionality is usually tested using an oxygen electrode as illustrated in textbook, Figure 11.9. If the mitochondria show high rates of oxygen consumption, high respiratory control ratios (>3), and ADP:O ratios close to the theoretical values (Web Table 11.1.A) they are in very good shape. Note that, for mitochondria with an appreciable alternative oxidase activity (measured in the presence of the cytochrome oxidase inhibitor, KCN), the respiratory control ratio and ADP:O ratio should be measured in the presence of an alternative oxidase inhibitor such as salicylhydroxamic acid (see Web Topic 11.2).

Mitochondrial Activities Can Be Determined In Situ

It is relatively straightforward to measure the activity of enzymes in the soluble matrix fraction isolated from mitochondria (see above). However, this fraction is very diluted compared to the matrix inside intact mitochondria (300–500 mg protein/ml!) and protein–protein interactions may have been lost. When measuring the activity of enzymes in fractions containing membranes—intact mitochondria and submitochondrial particles—a detergent can often be used to remove the permeability barrier that the inner membrane constitutes (e.g., Møller et al. 1996). However, it is also possible to use the ionophore alamethicin to permeabilize the inner membrane to molecules <1000 Da, which makes it possible to measure the activity of matrix and inner membrane enzymes in their native proteinaceous environment (Johansson et al. 2004).

Using alamethicin, it is even possible to measure the activity of cytosolic and mitochondrial enzymes inside intact cells since the ionophore also permeabilizes the plasma membrane (Matic et al. 2005).

References

Agius, S. C., Rasmusson, A. G., and Møller, I. M. (2001) NAD(P) turnover in plant mitochondria. Aust. J. Plant Physiol. 28: 461–470.

Boutry, M., Faber, A.-M., Charbonnier, M., and Briquet, M. (1984) Microanalysis of plant mitochondrial protein synthesis products. Plant Mol. Biol. 3: 445–452.

Day, D. A., Neuburger, M., and Douce, R. (1985) Biochemical characterisation of chlorophyll-free mitochondria from pea leaves. Aust. J. Plant Physiol. 12: 219–228.

Douce, R. (1985) Mitochondria in Higher Plants: Structure, Function, and Biogenesis. Academic Press, Orlando, FL.

Hausmann, N., Werhahn, W., Huchzermeyer, B., Braun, H.-P., and Papenbrock, J. (2003) How to document the purity of mitochondria prepared from green tissue of pea, tobacco and Arabidopsis thaliana. Phyton. 43: 215–229.

Johansson, F. I., Michalecka, A. M., Møller, I. M., and Rasmusson, A. G. (2004) Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilised plant mitochondria. Biochem. J. 380: 193–202.

Keech, O., Dizengremel, P., and Gardeström, P. (2005) Preparation of leaf mitochondria from Arabidopsis thaliana. Physiol. Plant. 124: 403-409.

Kruft, V., Eubel, H., Jänsch, L., Werhahn, W., and Braun, H.-P. (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol. 127: 1694–1710.

Matic, S., Geisler, D., Møller, I. M., Widell, S., and Rasmusson, A. G. (2005) Alamethicin permeabilises the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum) suspension cells. Biochem. J. 389(Pt3): 695-704.

Møller, I. M., Johansson, F., and Brodelius, P. (1996) Plant cell membranes. In Plant Membrane Biology: Proceedings of Phytochemical Society of Europe - 38, Møller, I. M. & Brodelius, P., eds, pp. 1–15. Clarendon Press, Oxford.

Møller, I. M., Lidén, A. C., Ericson, I., and Gardeström, P. (1987) Isolation of submitochondrial particles with different polarities. Methods Enzymol. 148: 442–453.

Struglics, A., Fredlund, K. M., Rasmusson, A. G., and Møller, I. M. (1993) The presence of a short redox chain in the membrane of potato tuber peroxisomes and the association of malate dehydrogenase with the membrane. Physiol. Plant. 88: 19–28.