From the
Post-translational import of precursor proteins into yeast
mitochondria is mediated by at least four protease-sensitive outer
membrane proteins: Mas20p, Mas22p, Mas37p, and Mas70p. These
``import receptors'' recognize either the N-terminal
targeting signal or some other feature of mitochondrial precursor
proteins. The only exception to this general rule appeared to be the
precursor to subunit Va of cytochrome c oxidase (COXVa).
Although this precursor carries a typical N-terminal mitochondrial
targeting sequence, its import into mitochondria has been suggested to
be independent of the known import receptors. Here we show that if
import into isolated yeast mitochondria is assayed under conditions in
which binding of the COXVa precursor to mitochondria is rate-limiting,
import is strongly inhibited by protease pretreatment of the
mitochondria or by antibodies against Mas20p. Post-translational import
of the COXVa precursor can thus proceed by the general,
receptor-mediated pathway.
One of the first steps of protein import into mitochondria is
the binding of precursor proteins to several ``import
receptor'' subunits on the cytosolic surface of the outer
membrane(1, 2) . There is currently no direct assay for
this step. Instead, binding to import receptors was initially inferred
from the facts that protein import was inhibited by gentle protease
treatment of intact mitochondria or by antibodies against
surface-exposed outer membrane
proteins(3, 4, 5, 6, 7, 8, 9) .
In more recent assays, precursors prebound to deenergized mitochondria
were chased into mitochondria upon reenergization of the organelles;
this ``productive binding'' was sensitive to the treatments
mentioned above(9, 10, 11, 12) .
However, these measurements are only valid if the receptor-mediated
step is rate-limiting for the import process. If this is not the case,
inhibition or activation of surface receptors may not inhibit precursor
import, since removal or inactivation of import receptors is rarely
quantitative and since the different receptor subunits may partially
substitute for each
other(6, 8, 9, 13, 14) .
COXVa
We have now
reinvestigated this problem under assay conditions in which encounter
of precursors with the mitochondrial surface is rate-limiting for
import. Such conditions are mandatory for assessing the action of
receptors in facilitating import of different precursor proteins. Our
results show clearly that import of the COXVa precursor is mediated by
trypsin-sensitive receptors, including Mas20p. Post-translational
import of the COXVa precursor into isolated yeast mitochondria can thus
occur by the usual, receptor-mediated import pathway.
The authentic precursors to F
Two reports have
suggested that import of the COXVa precursor into yeast mitochondria
does not occur via the receptor-mediated pathway (16, 17). These import
assays used mitochondrial concentrations equivalent to 0.5 mg/ml
protein. We readily confirmed these findings; under these assay
conditions, only the import of F
We thank Dr. Mike Cumsky for the plasmid
pDpT7-5a encoding COXVa, Sabine Rospert for the plasmid
SP6-HSP60, Klaus Pfanner and Mike Cumsky for discussion of this work,
Renate Looser and Hildegard Brütsch for technical assistance,
Verena Grieder, Margrit Jäggi, and Liselotte Müller for
photography, and members of the Schatz laboratory for critical reading
of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is made as a precursor with a typical
N-terminal basic, amphipathic targeting sequence composed of 20 amino
acids(15) . Import of the precursor into the mitochondrial inner
membrane uses the translocation channels of both mitochondrial
membranes (16) and the translocase function of mhsp70 (17) but has been reported to bypass the usual import receptors
on the mitochondrial surface. This receptor independence was inferred
from the observations that import of the COXVa precursor was not
inhibited by trypsin pretreatment of mitochondria (16) or by
genetically deleting the Mas70p or Mas20p receptor
subunits(17) . However, the published evidence suggested to us
that these import assays had been performed in the presence of a
functional excess of mitochondria and that binding of the precursor to
mitochondria might not have been rate-limiting.
Trypsin Pretreatment of
Mitochondria
Mitochondria from the yeast Saccharomyces
cerevisiae (strain D-273-10B, ATCC 25657) were isolated,
their amount was quantified spectroscopically at 280 nm in the presence
of SDS, and they were stored frozen as described
previously(18) . In the standard treatment, mitochondria (0.5
mg) were thawed, diluted into a final volume of 0.5 ml of import buffer
(20 mM HEPES-KOH, pH 7.4, 0.5 mM EDTA, 5 mM
MgCl, 0.6 M sorbitol, 2 mM KH
PO
, 25 mM KCl) containing 50
µg of trypsin, and incubated for 30 min on ice. Import buffer (1.0
ml) containing 1.0 mg/ml trypsin inhibitor was then added to
trypsin-treated or untreated mitochondria. After a further 5 min of
incubation, the mitochondria were isolated by centrifugation, washed
once in import buffer containing 0.1 mg/ml trypsin inhibitor and 1.0
mg/ml BSA, and finally resuspended in import buffer containing 1.0
mg/ml BSA.
In Vitro Import Assays
Trypsin-treated or
untreated mitochondria were diluted as indicated into 3.0 ml of import
buffer containing 1 mg/ml BSA, 0.5 mM ATP, and 0.5 mM NADH and incubated at the indicated assay temperature for 3 min.
Reticulocyte lysate containing the S-labeled precursor was
added, and at the indicated times, 0.5-ml samples were withdrawn to
microcentrifuge tubes containing 60 µl of a termination mix (30
µg of carrier mitochondria in import buffer containing 100
µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Proteinase K (4 µg)
was then added, and after incubation for 20 min on ice,
phenylmethylsulfonyl fluoride was added to 0.8 mM. The
mitochondria were isolated by centrifugation and solubilized in
SDS-sample buffer for electrophoretic analysis. Pretreatment of
mitochondria with IgGs specific for the major outer membrane protein
porin or for the receptor subunits Mas20p/Mas22p and Mas70p was as
described(6) .
Miscellaneous
The SalI-EcoRI DNA
fragment encoding the COXVa precursor protein was subcloned from
plasmid pDpT7-5a (16) into pSP64 (Promega) for
transcription with the SP6 RNA polymerase. SDS-PAGE was run on
Tris-Tricine-buffered gels (19).
and HSP60 as
well as the chimeric precursor SU9-DHFR are imported into yeast
mitochondria via protease-sensitive receptors on the mitochondrial
surface(6, 10, 12, 20, 21) .
Import is not absolutely dependent on these receptors but is slowed
5-10-fold if these receptors are removed or inactivated. Thus, it
is only possible to observe the influence of receptors on protein
import if binding of the precursor to the mitochondrial surface is
rate-limiting for the overall import process.
was strongly
inhibited by trypsin pretreatment of mitochondria (Fig. 1);
import of SU9-DHFR was less affected and that of HSP60 or COXVa was
completely unaffected.
Figure 1:
At 0.5 mg/ml mitochondria in the import
assay, trypsin pretreatment inhibits the import of only some
mitochondrial precursors. Mitochondria were pretreated with trypsin
(+) or left untreated (-) and then incubated at a final
concentration of 0.5 mg/ml mitochondrial protein with S-labeled precursors to F
(5 min at 25
°C), COXVa (5 min at 13 °C), HSP60 (5 min at 10 °C), or
SU9-DHFR (5 min at 25 °C). Import was measured by removing external
precursor with proteinase K and analyzing the mitochondria by SDS-PAGE,
fluorography of the dried gels, and quantification of the fluorograms
by PhosphorImager analysis. Photographs of the fluorograms are shown,
together with the quantitation expressed as a percentage of import into
mitochondria not treated with trypsin.
However, when we necessarily lowered the
amount of mitochondria in the in vitro assay and coimported
the precursors of F and COXVa, only the import of
F
was limited by the amount of mitochondria that had
been used in the assay referred to above; at 25 °C, the amount of
F
imported increased linearly with the concentration
of mitochondria in the assay at least up to 1.0 mg/ml (Fig. 2).
In contrast, import of COXVa was only dependent on the concentration of
mitochondria if that concentration was decreased about 20-fold and if
the assay temperature was lowered to 13 °C.
Figure 2:
Mitochondria limit the import rate of
COXVa only at lower concentrations and temperature. A mixture of the
precursors to F and COXVa was incubated with the
indicated amount of mitochondria for 5 min at 25 °C; import of the
precursor to COXVa was also tested for 5 min at 13 °C. Import was
analyzed as in Fig. 1.
The results of Fig. 2establish the proper assay conditions for testing the role
of mitochondrial receptors in the import of COXVa, since under these
conditions the encounter of the precursor with the mitochondrial
surface limits the rate of import. We therefore used these conditions
to retest mitochondria pretreated with trypsin for import of various
precursor proteins. Import of all precursors tested, including that of
COXVa, was now strongly inhibited by trypsin pretreatment (Fig. 3).
Figure 3:
Inhibition of COXVa import by trypsin
pretreatment of mitochondria. The import rate was measured for the
precursors to F (at 25 °C), COXVa (at 13 °C),
HSP60 (at 10 °C), or for SU9-DHFR (at 25 °C); the concentration
of mitochondria in the assay was 0.03 mg/ml, and samples were withdrawn
at the indicated times. Where indicated, the mitochondria had been
pretreated with 100 µg/ml trypsin. Import was analyzed as described
in Fig. 1.
Inhibition of COXVa import by trypsin pretreatment
of the mitochondria correlated with the degradation of the known import
receptor subunits (Fig. 4A). At low concentrations,
trypsin completely released Mas37p and Mas70p from the mitochondria,
clipped the C terminus of Mas20p, and slightly reduced the import of
COXVa. At higher trypsin concentrations, Mas20p and Mas22p were
completely degraded and COXVa import was severely inhibited.
Figure 4:
Import of COXVa is inhibited by
degradation of import receptor subunits with trypsin or by antibodies
against Mas20p. A, mitochondria were pretreated with the
indicated amounts of trypsin. One aliquot of treated and untreated
mitochondria was diluted to 0.03 mg/ml into import buffer and assayed
for import of F (5 min at 25 °C; hatchedbars), COXVa (2 min at 13 °C; blackbars), or SU9-DHFR (2 min at 25 °C; openbars). Another aliquot of treated and untreated
mitochondria (100 µg of mitochondrial protein) was analyzed by
SDS-PAGE and immunoblotting for the protease-sensitive import receptor
subunits Mas20p, Mas22p, Mas37p, and Mas70p, for the protease-resistant
import site protein Isp42p, and for the intermembrane space marker
cytochrome b
. A sample of mitochondria (MP) that had been treated with 100 µg/ml trypsin in a
hypoosmotic buffer to disrupt the outer membrane served as a control to
show that the outer membrane barrier had remained intact under the
conditions of trypsin treatment of mitochondria, protecting the highly
trypsin-sensitive intermembrane space protein cytochrome b
from the added protease (22). B,
mitochondria (10 µg) were pretreated in a final volume of 0.1 ml
with 100 µg of IgGs against porin, Mas20p, or Mas70p, diluted into
import buffer to a final concentration of 0.03 mg/ml, and assayed for
import of F
(5 min at 25 °C, hatchedbars), COXVa (2 min at 13 °C, blackbars), or SU9-DHFR (2 min at 25 °C, openbars).
Import
of COXVa was also inhibited by pretreatment of mitochondria with IgGs
against the import receptor subunit Mas20p (Fig. 4B).
IgGs against the Mas70p receptor subunit had no effect. As pointed out
here and earlier(11) , a lack of inhibition in import assays
cannot be reliably interpreted, and we do not conclude that import of
COXVa bypasses the function of Mas70p. However, we do conclude that the
Mas20p subunit is involved and that import of the COXVa precursor, like
that of all other precursors that use the general import machinery, is
initiated by the protein import receptor.
-ATPase and dihydrofolate
reductase; HSP60, heat shock protein 60; F
,
-subunit of the F
-ATPase; BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.