Biomedical Engineering Reference
In-Depth Information
6. Initiate import reaction by addition of precursor to sample #5
(longest incubation period) and subsequent precursor addition
to samples #4-1 (with #1 = shortest incubation period). Mix
after addition by fl icking the tubes.
7. Stop import reactions according to time scheme by addition of
1× valinomycin from 100× stock and moving samples on ice.
8. Prepare six fresh tubes (samples #7-12) with 1
ʼ
l PK (f.c.:
50
ʼ
g/ml) in 50
ʼ
l SEM buffer.
9. Add 50
l each of samples #1-6 to PK-containing samples
#7-12 and incubate for 15 min on ice; add 50
ʼ
ʼ
l SEM buffer
to samples #1-6 and likewise keep on ice.
10. Stop PK treatment by addition of 1 mM PMSF from 0.1 M
stock (
see
Note 5
); for equal treatment of all samples, add
PMSF to non-PK-treated samples as well.
11. Keep samples on ice for other 10 min, before pelleting mito-
chondria by centrifugation at 12,000 ×
g
for 10 min.
12. Remove supernatants carefully, wash mitochondrial pellet by
addition of 200
l SEM buffer containing 1 mM PMSF, and
centrifugation at 12,000 ×
g
for 8 min.
13. After removal of supernatants denature mitochondria in 1×
Laemmli buffer.
ʼ
The import of precursor proteins in a denatured state can be useful
for the analysis of mitochondrial import defects in more detail.
Denaturation of the preprotein to be imported facilitates its import,
as the import reaction is no longer dependent on the functionality
of the mtHsp70 system for unfolding. ATP-driven precursor
unfolding is of particular importance for the import of tightly
folded proteins or protein domains. Thus, comparison of the
import effi ciencies of both natively folded and denatured preprot-
eins in mitochondrial preparations from different strains can have
implications both for the tertiary structure of the preprotein
imported, and for the mtHsp70 protein (un)folding capacity.
3.4.1 Import of Urea-
Denatured Precursor
Proteins
1. Thaw the preprotein to be imported on ice.
2. Add three volumes of cold-saturated NH
4
(SO
4
)
2
and mix by
vortexing at low speed.
3. Incubate on ice for 30 min with occasional vortexing.
4. Centrifuge precipitates down at 20,000 ×
g
for 15 min.
5. Aspirate supernatant carefully, and resuspend protein pellet in
the required volume of urea buffer by shaking at room tem-
perature on a thermomixer at maximum speed for 15 min.
6. Pellet non-denatured protein material by centrifugation at
20,000 ×
g
for 5 min.
