Materials and Reagents

Candida albicans culture
Tris-SO₄
DTT
Sorbitol
Lyticase (Sigma-Aldrich, catalog number: L2524 )
K⁺MES [Potassium 2-(N-morpholino)ethanesulfonate]
K⁺HEPES [Potassium 4-(2-hydroxyethyl)-1-piperazineethanesulfonate]
PMSF
Tris-DTT buffer (see Recipes)
Sorbitol buffer (see Recipes)
KH₂PO₄ & K₂HPO₄ (see Recipes)
Breaking Buffer (BB) 6.0 (see Recipes)

Equipment

Incubator shaker with temperature control
Centrifuges
Dounce (tight dounce 40 ml) (Wheaton, catalog number: 06-435C )

Procedure

Culture preparation
Inoculate with sufficient starter culture to produce a culture of Candida albicans cells with an OD₆₀₀= 1.0-2.0 (an OD₆₀₀ of up to 7 has worked in our hands) at the desired start time the next morning. Shake at 200 rpm at 30 °C overnight.
Method
1. Collect cells by spinning at 4,000 x g/10 min/RT (room temperature).
2. Pour off medium
3. Resuspend cells in dH₂O in weighed centrifuge tubes.
4. Spin at 2,500 x g/5 min/RT. Reweigh to get mass of cells.
5. Resuspend cells in Tris-DTT buffer (see Recipes) ~5 ml per g of cells.
6. Incubate for 15 min at 30 °C with gentle shaking (~100 rpm).
7. Spin at 2,500 x g/5 min/RT.
8. Resuspend pellet in ~5 ml/g of cells pre-warmed 1.2 M sorbitol buffer.
9. Spin at 2,500 x g/5 min/RT.
10. Weigh out 0.2 mg/g of cells of lyticase. Dissolve in 2 ml pre-warmed (30 °C) 1.2 M sorbitol buffer per gram of cells. Resuspend pellet in this solution.
11. Incubate cells in lyticase solution for ~60 min/30 °C with gentle shaking (~100 rpm) or until spheroplasts form (check spheroplast formation by osmotic shock: Add 30 μl cells to 2 ml 1.2 M sorbitol and water. After vortexing water sample should go clear).
12. Spin down for 2,500 x g/5 min/RT. Discard supernatant.
13. Resuspend in cold 1.2 M sorbitol buffer ~5 ml/g of cells. Spin 2,500 x g/5 min/4 °C.
14. Resuspend in minimal amount of cold breaking buffer (BB) 6.0 then make up in BB 6.0 containing 1 mM PMSF final (~4 ml/g cells)
15. Homogenize 15 times using a tight dounce. Dounce should be ~3/4 full or a bit less. Up stroke must be fast and steady. Bubbles can break mitochondria so try not to let it pop out.
16. Spin homogenate 5 min/4 °C/3,000 x g. Save supernatant in a new tube and keep on ice (supernatant should be cloudy).
17. (Optional step for higher yield) Resuspend pellet again in BB 6.0 with PMSF and tight dounce and spin as above. Combine supernatants. Discard pellets.
18. Spin combined supernatants 5 min/4 °C/3,000 x g. Save supernatant (repeat this spin step to clear more of the contaminating membranes if still getting a large pellet).
19. Spin supernatant for 10 min/4 °C/12,000 x g.
20. Pour off the supernatant and resuspend pellet in a small amount of BB 7.4 (with bovine serum albumin if mitochondria are to be used for in vitro import assays).
21. Spin down 10 min/4 °C/12,000 x g then remove any white membranes that surround reddish-brown mitochondrial pellet before resuspending in a minimal amount of BB 7.4 with BSA.
22. Repeat spin step above if still contaminating membranes present.
23. Estimate the mitochondrial concentration as follows: Add 10 μl crude mitochondria to 990 μl 0.6% SDS. As a blank add 10 μl BB 7.4 with BSA to 990 μl 0.6% SDS. Measure A₂₈₀ using Quartz cuvette. An absorbance value of 0.21 corresponds to 10 mg/ml protein in the undiluted mixture.
24. Aliquot at appropriate volumes and snap freeze in dry ice/liquid nitrogen.

Recipes

Tris-DTT
0.1 M Tris-SO₄ (pH 9.4)
10 mM DTT (make fresh from frozen 1 M DTT aliquots just before use)
1.2 M Sorbitol buffer
1.2 M sorbitol
20 mM KPi (pH 7.4)
Breaking buffer, BB (6.0)
0.6 M sorbitol
20 mM K⁺MES (pH 6.0) (add PMSF just prior to use to 1 mM final concentration)
PMSF
34 mg/ml (0.2 M) in ethanol
Breaking buffer, BB (7.4)
0.6 M sorbitol
20 mM K⁺HEPES (pH 7.4) (adjust pH with KOH)
2.4 M sorbitol stock (aqueous solution deionised)
1 M K⁺MES stock (pH 6.0)
Filter sterilize and store in foil at RT
1 M KPi (pH 7.4)
Make from stocks of 1 M KH₂PO₄ and stock of 1 M K₂HPO₄ and mix them until pH = 7.4. Do not titrate with HCl or NaOH.

Acknowledgments

The protocol was adapted from: Hewitt et al. (2012). The work in the Traven lab and Lithgow lab on Candida albicans mitochondria is supported by a project grant from the Australian National Health and Medical Research Council (APP1023973). V.H. is the recipient of an Australian Postgraduate Award.

References

Daum, G., P. C. Bohni, et al. (1982). Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257(21): 13028-13033.
Hewitt, V. L., Heinz, E., Shingu-Vazquez, M., Qu, Y., Jelicic, B., Lo, T. L., Beilharz, T. H., Dumsday, G., Gabriel, K., Traven, A. and Lithgow, T. (2012). A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways. Proc Natl Acad Sci U S A 109(49): E3358-3366.
Hewitt, V., Lithgow, T. and Traven, A. (2013). Candida albicans Mitochondrial Protein Import Assay. Bio-protocol 3(4): e331.