Import of Proteins into Isolated Yeast Mitochondria - Membrane Trafficking - page 43

5. Collect the cells by centrifugation at 2,000 × g for 5 min.
Discard the supernatant and wash the cell pellet with sorbitol
buffer (5 mL/g of wet weight).
6. Collect the cells again by centrifugation at 2,000 × g for 5 min.
Discard the supernatant.
7. Resuspend the cells in MP2 buffer (6.7 mL/g of wet weight).
8. Add the Zymolyase 20T (2 mg/g of wet weight) to the
suspension and incubate under agitation (approx. 140 rpm)
for 20-40 min at 30 °C until spheroplasts have formed
( see Note 16 ).
9. All following steps will be performed on ice using ice-cold
buffers, keeping the samples always on ice and centrifugation
steps performed at 4 °C.
10. Harvest the spheroplasts by centrifugation at 2,000 × g
for 5 min.
11. Resuspend the pellet in MP3 buffer (6.7 mL/g of wet weight)
by gently shaking or stirring with a pipette.
12. Transfer the suspension to a 50 mL Dounce homogenizer
(tight-fitting glass pestle) and homogenize with 15 strokes.
13. Dilute the resulting homogenate with MP3 buffer (again
6.7 mL/g of wet weight).
14. Centrifuge the homogenate at 2,000 × g for 5 min to spin
down cell debris. Keep the supernatant and transfer it to a fresh
centrifugation bucket.
15. Centrifuge at 17,000 × g for 12 min. Discard the supernatant.
16. Resuspend the pellet carefully in 10 mL of SH or SEH buffer.
17. Centrifuge at 17,000 × g for 12 min. Discard the supernatant.
18. Resuspend the pellet in 0.5 mL of SH or SEH buffer.
19. Take an aliquot to determine protein concentration.
20. Dilute the mitochondrial suspension to a final concentration of
10 mg/mL in SH buffer.
21. Make aliquots of 30-50 ʼL, immediately snap-freeze in liquid
nitrogen. Store at −80 °C.

3.3 Synthesis
of Preproteins

1. To make 50 ʼL of the transcription reaction, mix 30 ʼL of the
premix solution, 2.2 ʼL RNasin, 2.5 ʼL m 7 G-cap, 0.8 ʼL SP6
RNA polymerase, and 15 ʼL of the DNA template ( see Note 17 ).
2. Incubate for 1 h at 37 °C.
3. Add 5 ʼL LiCl and 150 ʼL ethanol (−20 °C) in order to pellet
the RNA.
4. Incubate for 15 min at −20 °C.
5. Spin down the RNA at 35,000 × g and 4 °C for 30 min.

3.3.1 In Vitro
Transcription