Import of Proteins into Isolated Yeast Mitochondria - Membrane Trafficking - page 44

6. Wash the RNA pellet with 70 % ethanol (−20 °C).
7. Spin down the RNA at 35,000 × g and 4 °C for 5 min.
8. Remove ethanol completely at room temperature. Resuspend
the RNA pellet in 30 ʼL water and add 0.8 ʼL RNasin.
9. Make single-use aliquots and store at −80 °C ( see Note 18 ).

1. To make an in vitro translation reaction, mix 140 ʼL rabbit
reticulocyte lysate, 4 ʼL of the amino acid mixture minus
methionine, 4 ʼL RNasin, 16 ʼL [ 35 S]-Met, and 30 ʼL RNA
from the transcription reaction.
2. Incubate for 1 h at 30 °C.
3. Chase the reaction by adding 8 ʼL of cold methionine.
4. Incubate for 5 min at 30 °C.
5. Make single-use aliquots (12 ʼL) of the lysate. Freeze in liquid
nitrogen and store at −80 °C.
6. To assess the translation product, analyze 1 ʼL of the lysate by
SDS-PAGE and autoradiography ( see Note 19 ).

3.3.2 In Vitro Translation

3.4 In Vitro Import
of Preproteins into
Isolated Mitochondria

The standard import protocol monitors the translocation of pre-
proteins across the outer mitochondrial membrane by assessing
their resistance against externally added proteases. In addition, the
dependency of the import reaction on ATP or the membrane
potential can also be tested ( see Notes 20 - 22 ). To localize the
imported proteins after the import reaction, the mitochondria can
be converted to mitoplasts, i.e., mitochondria with the outer mem-
brane ruptured by hypotonic swelling. Upon addition of protease,
proteins that have expose domains in the IMS can be degraded by
proteases, whereas proteins topologically confined to the matrix
will remain intact.

1. Prepare six tubes with 50 ʼL 2× import buffer.
2. Add in tubes 1-3: 42 ʼL water, 1 ʼL ATP, 1 ʼL NADH, and
5 ʼL of mitochondria ( see Note 23 ).
3. Add in tubes 4-6: 43 ʼL water, 1 ʼL VAO buffer, and 5 ʼL of
mitochondria.
4. Incubate all tubes for 5 min at 25 °C ( see Note 24 ).
5. Add 1 ʼL of the radioactive protein lysate to all six tubes
( see Note 25 ).
6. Incubate for 20 min at 25 °C.
7. Add 900 ʼL SH buffer to tubes 1, 2, 4, and 5. Add 900 ʼL
HEPES/KOH pH 7.4 to tubes 3 and 6. Add 10 ʼL of PK to
tubes 2, 3, 5, and 6 ( see Note 26 ).
8. Incubate for 30 min on ice.