Mitochondrial import depends on ATP. By using an uncoupler like CCCP, we can find out if the protein is just bound to the outer membrane or is really imported and processed.
If imported, the protein will protease protected. Trypsin is used to do this control. Set up microcentrifuge Tubes - you need four tubes for each plasmid
Tube#
1
2
3
4
Mitochondria
100
100
100
100
Extra lysate
48
48
48
48
Translation
2
2
2
2
CCCP
--
+
--
+
Trypsin
--
--
+
+
PMSF
--
--
+
+
SBTI
--
--
+
+
Incubate at 30Incubate at 30°C for one hour with occasional mixing.
Remove tubes after incubation and keep on ice.
To tubes 3&4 add 2.3ml of 1 mg/ml trypsin. Leave on ice for 30 min.
Add 3 ml of 100mM PMSF and 4.6ml of 10 mg/ml soybean trypsin inhibitor (SBTI).
Incubate on ice for 10 min.
Centrifuge at 4°C for 10 min. Full speed in a microcentrifuge.
Remove supernatant and resuspend pellet in 150ml of medium (or resuspending buffer) using a yellow tip.
Centrifuge at 4°C for 10 min. and repeat the washing procedure once more.
Finally add 20ml of 2x gel sample buffer and sonicate briefly to dissolve the mitochondrial pellet.
Boil briefly, then run 10ml on mini gels.
As a control use: UCP (uncoupling protein) 32kDa, imports into mitochondria quite efficiently. Alternatively, pOCTgPA is also imported into mitochondria and has a cleaved amino-terminal import sequence.
A typical experiment will give the following results:
Tube #
Protein bound and imported into mitochondria.
Protein only bound to mitochondria- CCCP inhibits import.
Protein imported to mitochondria- trypsin will digest any bound but not imported protein.
No protein- CCCP inhibits import, trypsin digests bound protein.
