Biomedical Engineering Reference
In-Depth Information
7. Transfer supernatant to a fresh tube and keep on ice until use.
8. Perform import reaction essentially as described in
Subheading
3.4
.
1. Run samples on a sodium dodecyl sulfate polyacrylamide
(SDS-PAGE) gel with a resolving gel acrylamide concentration
of 12 %. Always run three control lanes with 1, 2 and 5 % of the
preprotein amount used in the assay for quantifi cation.
2. Transfer proteins to a Western Blot membrane (e.g.,
Millipore
).
3. Immunodecorate with antibodies directed against mouse
DHFR.
4. Detect signals using peroxidase-coupled secondary antibodies
and enhanced chemiluminescence.
5. Evaluate raw data with image analysis software (
see
Subheading
3.4.1
).
3.5 Analysis
of the Import Reaction
Evaluation of import kinetics can be done by quantifi cation of
protein bands using standard image analysis software (i.e., ImageJ)
provided that the signals have not saturated. Band intensities in
samples not treated with protease provide information on process-
ing effi ciencies, while determination of the effi ciency of complete
import is done from the protease-treated samples. Use the control
lanes as references for protein amounts.
The ligand methotrexate (MTX) stabilizes the tertiary structure of
the DHFR domain and thereby prevents translocation of fusion
proteins containing a C-terminal DHFR domain across the mito-
chondrial membranes [
11
,
12
]. When b
2
(
3.6 Dissection
of a Functional Import
Intermediate on Blue
Native PAGE
)-DHFR is imported in
the presence of MTX, it accumulates in a translocation intermedi-
ate that spans both mitochondrial membranes. Determination of
the constituents of this complex by Blue Native gel electrophoresis
(BN-PAGE) revealed the presence of TOM-components, as well as
the core components of the TIM23 complex of the inner mem-
brane. The translocase complexes are connected to each other by
the preprotein in transit resulting in the formation of a large mul-
tiprotein complex comprising both outer and inner membrane
components [
9
,
13
]. Formation of the TOM-TIM-supercomplex
is dependent on the membrane potential across the inner mem-
brane, and results in an almost quantitative shift of the TIM23 core
subunits from a size of ~90 kDa to a size of ~600 kDa, on BN-PAGE
(
see
Fig.
4a
).
The accumulation of b
2
(167)
ʔ
-DHFR in import sites is achieved
by pre-incubation of the preprotein in the presence of 5
ʔ
M MTX,
and addition of MTX in the import reaction, which results in the
stable arrest of the preprotein inside the import channels.
Formation of the supercomplex can be monitored by Western
blot using anti-Tim23 antiserum. BN-PAGE is specifi cally suited
ʼ
