(Received for publication, October 21, 1994; and in revised form, December 8, 1994)
From the
Protein import into yeast mitochondria is mediated by the four outer membrane receptors Mas70p, Mas37p, Mas20p, and Mas22p. These receptors may function as two subcomplexes: a Mas37p/Mas70p heterodimer and an acidic complex consisting of Mas20p and Mas22p. To assess the relative contribution of these subcomplexes to precursor binding, we allowed different precursors to bind to the surface of deenergized mitochondria, then reenergized the mitochondria and measured the chase of the bound precursors into the organelles. Productive binding of several precursors with a positively charged amino-terminal matrix targeting sequence, such as SU9-DHFR, hsp60, and mitochondrial cpn10, was strongly inhibited by salt, by low concentrations of a mitochondrial presequence peptide, and by a deletion of Mas20p, but was independent of Mas37p/Mas70p. In contrast, productive binding of the ADP/ATP carrier was not inhibited by salt, the presequence peptide, or a deletion of Mas20p, but was strongly dependent on Mas37p/Mas70p. The precursors of alcohol dehydrogenase III and the Rieske iron-sulfur protein had binding properties between these two extremes. The productively bound precursor of cpn10 could be cross-linked to Mas20p. We conclude that Mas20p binds mitochondrial precursor proteins through electrostatic interactions with the positively charged presequence, whereas Mas37p/Mas70p may recognize some feature(s) of the mature part of precursor proteins.
Import of proteins into mitochondria requires the specific
recognition of cytoplasmically synthesized mitochondrial precursor
proteins by receptor proteins in the mitochondrial outer
membrane(1, 2) . Studies with Saccharomyces
cerevisiae and Neurospora crassa have identified three
such receptors: a 20-kDa protein termed Mas20p in yeast and MOM19 in N. crassa(3, 4) , a 70-kDa protein termed
Mas70p in yeast and MOM72 in N. crassa(5, 6, 7) , and a 22-kDa protein
termed Mas22p in yeast and MOM22 in N.
crassa(8, 9) . A fourth receptor, a 37-kDa
protein termed Mas37p, has so far been identified only in yeast. ()These four receptors exist as two hetero-oligomeric
subcomplexes that may loosely interact with each other: a Mas37p/Mas70p
heterodimer and an oligomer containing Mas20p and
Mas22p(
)(10) .
The relative contributions of
these different receptor subunits to the overall import process have
not been firmly established. Import experiments with isolated yeast
mitochondria have suggested that different subsets of precursors differ
in their dependence on each of the receptor subcomplexes. Mas37p/Mas70p
promotes the import of the ADP/ATP carrier, cytochrome c, the F
-ATPase
-subunit, and the
mitochondrial isozyme III of the alcohol dehydrogenase, but not import
of artificial precursors containing dihydrofolate reductase (DHFR) (
)as a passenger protein. In contrast, Mas20p/Mas22p
interact with most or all precursors containing mitochondrial
presequences, including DHFR-containing fusion
proteins(4, 7, 11, 12) . However,
only Mas22p is essential for protein import and viability of yeast;
Mas20p, Mas37p, and Mas70p can be deleted singly or in pairwise
combination without blocking import as long as the level of Mas22p is
maintained(9) .
It is still unknown how the import receptors specifically recognize precursor proteins destined for mitochondria. Experiments in which the presequences of authentic precursors were fused to DHFR have suggested that Mas20p/Mas22p, through their cytosolically exposed ``acid bristles,'' may bind the basic and amphiphilic mitochondrial presequences(9, 12) . Recognition by Mas37p/Mas70p appears to be more complex, involving a precursor's ``mature'' part(11) .
In this
study we have attempted to determine which mitochondrial receptors
specifically recognize NH-terminal mitochondrial
presequences. We found that the NH
-terminal basic and
amphiphilic presequences are bound by the acid bristle complex
containing Mas20p and Mas22p, that this binding is blocked by a
chemically synthesized presequence peptide, and that it is
predominantly mediated by a salt-sensitive electrostatic interaction.
In contrast, binding of precursors to Mas37p/Mas70p is only partly
inhibited by high salt or by a presequence peptide, confirming that
this receptor subcomplex recognizes determinants in the mature part of
precursor proteins.
We chose
the following precursors: a fusion protein containing the first 69
amino acids of the N. crassa FF
-ATPase
subunit 9 precursor fused to mouse DHFR (SU9-DHFR)(18) ,
mitochondrial hsp60, the mitochondrial isozyme of alcohol dehydrogenase
III (pADHIII), the Rieske Fe/S protein, and the ADP/ATP carrier (AAC).
These precursors were synthesized by coupled transcription/translation
in a reticulocyte lysate and were used without further purification.
Some experiments were also performed with chemically pure,
radioiodinated yeast mitochondrial cpn10. This protein has an
amphiphilic NH
-terminal matrix-targeting signal that is not
cleaved upon import(15) .
When the radiolabeled precursors shown in Fig. 1A were incubated with deenergized mitochondria in a low-salt buffer, 10-30% of the added precursor molecules bound to the mitochondrial surface: the precursors remained accessible to added protease, and those with a cleavable presequence remained unprocessed. Upon a subsequent chase, between 25 and 50% of the prebound precursors could be chased into the mitochondria: they became protease-inaccessible, and any cleavable presequence was removed (Fig. 1A). Thus, between 25 and 50% of the bound precursor molecules were productively associated with the mitochondrial surface (11) .
Figure 1:
Precursor binding to the mitochondrial
surface. A, different precursors can be bound productively to
the mitochondrial surface. The precursors of SU9-DHFR, hsp60, pADHIII
and the ADP/ATP carrier (AAC) were translated in vitro and incubated for 5 min at 0 °C with isolated mitochondria in
buffer A containing CCCP. The mitochondria were reisolated and washed.
To analyze binding, the mitochondrial pellet was resuspended in buffer
A containing CCCP and left on ice (- chase). For the
chase reaction the mitochondria were reenergized and chased at 25
°C for the indicated times in min. The chase reaction was stopped
by addition of valinomycin to 0.1 µg/ml. Where indicated (+ Prot. K), surface-bound precursor was digested with
proteinase K, followed by addition of PMSF to 1 mM. The
mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the precursor added to the assay; p and m, precursor or mature form of a protein,
respectively. B, the surface-bound precursor of SU9-DHFR
contains a tightly folded DHFR domain. The in vitro translated
precursor of SU9-DHFR was bound to deenergized mitochondria; the
mitochondria were reisolated, washed, and either resuspended in buffer
A containing CCCP (Bind) or reenergized and chased for 20 min
at 25 °C (Chase). The chase reaction was stopped by
addition of valinomycin to 0.1 µg/ml. To analyze the folding status
of the bound precursor, the mitochondria containing the bound precursor
were reisolated and treated with proteinase K, followed by inactivation
of the protease with 1 mM PMSF (+PK). The
supernatant (Sup) was precipitated with trichloroacetic
acid(30) . All samples were analyzed by SDS-PAGE and
fluorography. pSU9-DHFR and mSu9-DHFR, uncleaved
(``precursor'') and cleaved (``mature'') form of a
fusion protein containing the first 69 amino acids of the N. crassa FF
-ATPase subunit 9 precursor fused to
mouse DHFR; DHFR*, folded, protease-resistant DHFR
domain.
The DHFR domain of artificial precursors containing DHFR as a passenger protein is tightly folded, even after synthesis in vitro, but must be at least partly unfolded during import into mitochondria (19) . In order to determine whether unfolding is mediated by the mitochondrial receptors, SU9-DHFR was bound to uncoupled mitochondria. The mitochondria were then reisolated and divided into three aliquots. The first aliquot was analyzed directly for bound precursor (Fig. 1B, Bind - PK). The second aliquot was chased and then treated with protease in order to verify that the precursor had bound productively (Fig. 1B, Chase + PK). The third aliquot was treated with protease without a chase in order to check for release of the protease-resistant DHFR ``core'' fragment that is generated only from folded DHFR(19) . As the bound precursor was quantitatively converted to this core fragment (DHFR*; Fig. 1B, Sup + PK), its DHFR domain must have remained folded, only the presequence being exposed to the protease.
In the experiments summarized in Fig. 2, the different precursors were bound to deenergized mitochondria in the presence of varying salt concentrations. The mitochondria were then reisolated, washed, and chased in low-salt buffer. Different aliquots of the mitochondria were then analyzed for productively bound precursor as outlined in Fig. 1. Adding 50-200 mM NaCl to the binding buffer inhibited productive binding of pSU9-DHFR and phsp60 almost completely (Fig. 2, A and C), but had little effect on productive binding of AAC (Fig. 2, B and C). Productive binding of the precursors of ADHIII (Fig. 2, B and C) and the Rieske Fe/S protein (not shown) showed an intermediate salt sensitivity. Thus, addition of 100 mM NaCl lowers the binding of ADHIII 3-4-fold.
Figure 2: Productive binding of the precursors of Su9-DHFR and of hsp60 is highly salt-sensitive. The precursors of SU9-DHFR and hsp60 (A) and those of ADHIII and the ADP/ATP carrier (AAC) (B) were translated in vitro and incubated with deenergized mitochondria in buffer A containing the indicated concentrations of NaCl at 0 °C for 5 min. The mitochondria were reisolated and washed. They were then either resuspended in buffer A containing CCCP and kept on ice (- chase) or suspended in buffer A, reenergized, and chased at 25 °C for 20 min (+ chase). Surface-bound precursor was digested with proteinase K followed by addition of PMSF to 1 mM. The mitochondria were reisolated and analyzed by SDS-PAGE, fluorography, and densitometric quantification of the bands. 20% STD, 20% of the precursor added to each assay. p and m, precursor or mature form, respectively, of each protein. C, densitometric quantification of the results shown in A and B (+ chase). For calculating chase efficiencies, the amount of precursor chased in the absence of added NaCl was taken as 100%.
Complete inhibition of productive binding of pSU9-DHFR and phsp60 is
also seen upon addition of 35 mM NaSO
instead of 100 mM NaCl, suggesting that the extent of
inhibition is dependent on the ionic strength of the binding buffer
(data not shown). An earlier report had suggested that pSU9-DHFR forms
little, if any, productive binding intermediate(18) . This
result can now be explained by the fact that binding and chase had been
measured in a buffer containing 80 mM KCl; according to our
results, this salt concentration inhibits productive binding of
pSU9-DHFR by more than 90%.
Figure 3: Import of SU9-DHFR into mitochondria is also salt-sensitive. The precursors of SU9-DHFR and ADP/ATP carrier (AAC) were translated in vitro and incubated with fully energized mitochondria in the presence of different concentrations of NaCl at 15 °C for the indicated times (in minutes). Nonimported precursor was digested with proteinase K followed by addition of PMSF to 1 mM. The mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the precursor added to each assay. p and m, precursor or mature forms, respectively, of the respective protein.
Figure 4:
Productive binding of SU9-DHFR to the
mitochondrial surface is predominantly mediated by Mas20p. The
precursors of SU9-DHFR and the ADP/ATP carrier (AAC) were
translated in vitro and incubated as outlined in the legend of Fig. 2with deenergized mitochondria from wild-type cells (A), from a mutant lacking both Mas37p and Mas70p (70
37) (B), or
20 res
from a mutant lacking Mas20p, but with normal levels of Mas22p (C). The samples were chased as described in the legend to Fig. 2A and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the amount of precursor added to the assay. p and m, precursor or mature forms, respectively, of
each protein.
Productive binding of SU9-DHFR to yeast mitochondria is thus predominantly mediated by Mas20p and independent of Mas37p/Mas70p. The Mas20p contains a highly acidic cytosolic domain that could bind positively charged presequences(4) .
Figure 5: Productive binding of SU9-DHFR to the mitochondrial surface is specifically inhibited by the presequence peptide of mammalian hsp60. A, amino acid sequences of the chemically synthesized prepeptide of hsp60 from rat liver mitochondria (pre-hsp60) and of the basic hydrophilic peptide synB2. B, the in vitro translated precursors of SU9-DHFR (pSU9-DHFR) and of the ADP/ATP carrier (AAC) were bound to deenergized mitochondria in the presence of the indicated concentrations of the hsp60 prepeptide (pre-hsp60) or of the hydrophilic peptide synB2(24) . The mitochondria were reisolated, washed, and reenergized. Bound precursor was then chased into the mitochondria at 25 °C for 20 min. Nonimported precursor was digested with proteinase K followed by addition of 1 mM PMSF, and the mitochondria were reisolated and analyzed by SDS-PAGE and fluorography. 20% STD, 20% of the total precursor added to each assay. C, quantification of the results shown in B. For calculating chase efficiencies, the amount of precursor chased in the absence of added prepeptide was taken as 100%.
Figure 6:
Productive binding of the chemically pure
precursor of yeast mitochondrial chaperonin 10 to the mitochondrial
surface is highly salt-sensitive. A, productive binding of
purified chaperonin 10 (cpn10). Two-hundred ng of I-labeled chemically pure yeast mitochondrial cpn10
(approximately 4
10
cpm/mg of protein) were bound
to deenergized mitochondria. The mitochondria were then subjected to a
chase for the indicated times (in minutes), treated with proteinase K
to digest nonimported precursor, and analyzed by SDS-PAGE and
autoradiography. B, productive binding of purified cpn10 is
highly salt-sensitive. Two-hundred ng of
I-labeled cpn10
was bound to isolated mitochondria in buffer A containing the indicated
concentration of NaCl for 5 min at 0 °C. The samples were then
treated as described in Fig. 2and analyzed by SDS-PAGE,
autoradiography, and densitometric quantification of the
autoradiograms. C, quantification of the results shown in B. The amount of precursor chased in the absence of added NaCl
is taken as 100%.
Figure 7:
The productively bound chemically pure
precursor of mitochondrial chaperonin 10 can be specifically
cross-linked to the import receptor Mas20p. I-Labeled
chemically pure precursor of yeast mitochondrial cpn10 (200 ng; 4
10
cpm/mg of protein) was bound to isolated
mitochondria in CCCP-containing buffer A for 5 min at 0 °C. The
mitochondria were reisolated, washed, and divided into two equal
aliquots. The first aliquot was resuspended in buffer A containing CCCP (- chase); the second aliquot was chased at 25 °C
for 20 min. Each of the two aliquots was then divided further into two
parts; one part was treated for 30 min at 0 °C with 200 µM of the homobifunctional cross-linker disuccinimidyl suberate (DSS), followed by addition of Tris/Cl (pH 7.4) to 0.2 M. The other part was left untreated. All four mitochondrial
samples were then reisolated and either analyzed directly by SDS-PAGE
and autoradiography or subjected to immunoprecipitation (I.P.)
with antisera monospecific for either Mas20p, Mas70p, or porin followed
by SDS-PAGE and autoradiography. M.W., protein molecular mass
standards. *A, cross-linked
product.
The productively bound
precursor of mitochondrial cpn10 is thus directly bound to the receptor
subunit Mas20p. As cpn10 is bound via its NH-terminal
targeting sequence (see above), this result strongly suggests that
mitochondrial targeting sequences are recognized by the acidic
Mas20p/Mas22p receptor subcomplex.
The present study shows that the basic and amphiphilic
targeting sequence at the NH terminus of a mitochondrial
precursor protein is sufficient for recognition of the precursor by the
acidic mitochondrial import receptor Mas20p. This result directly
correlates a property of a mitochondrial targeting sequence with a
component of the mitochondrial protein import machinery. The
cytosolically exposed acid bristles of Mas20p and its partner subunit
Mas22p are uniquely suited to bind the exposed basic mitochondrial
targeting signals. This interaction is highly salt-sensitive and thus
is largely mediated by electrostatic interactions. However,
amphiphilicity of the targeting signal must also be important, because
precursor binding is not inhibited by a basic peptide which is not
amphiphilic.
The cross-linking data and the binding experiments with
Mas20p-deficient mitochondria show that precursors with an
NH-terminal presequence are recognized by Mas20p, but these
data do not exclude the possibility that presequences are also
recognized by other receptor subunits. An additional subunit that
probably recognizes presequences is Mas22p, which is tightly bound to
Mas20p.
The identification of Mas20p as a
``presequence receptor'' does not imply a hierarchy of
receptor function nor does it suggest that Mas20p is a ``master
receptor''. On the contrary, our present data strengthen the view
that the mitochondrial receptor subunits cooperate with one other as a
functional unit in which the different subunits recognize different
parts of a precursor protein (Fig. 8). For example, the
precursors of ADHIII, the F-ATPase
-subunit, or the
Rieske Fe/S protein probably bind simultaneously to several receptors:
to Mas20p/Mas22p mainly via the presequence and to Mas37p/Mas70p mainly
via the mature part. As a result, productive binding of these
precursors is less sensitive to salt or presequence peptide.
Figure 8:
Cooperation of receptor subunits in
binding precursors to the yeast mitochondrial surface. Precursor
binding is mediated by at least four distinct receptor subunits. These
subunits exist as two hetero-oligomeric complexes that loosely interact
with one another. Mas20p (possibly in association with Mas22p) binds
mitochondrial precursor proteins through electrostatic interactions
with the positively charged presequence, whereas the Mas70p/Mas37p
receptor subcomplex interacts with the mature domain of the precursor
molecule. OM, mitochondrial outer membrane; + and
-, positively and negatively charged amino acids; dots between the soluble domains of Mas20p and Mas70p indicate a weak
interaction between these receptor domains as suggested by studies
using the two hybrid system.
Although Mas20p is a presequence receptor, several presequence-containing precursors are efficiently recognized by Mas37p/Mas70p. However, Mas37p/Mas70p differs from Mas20p in that it can only recognize precursors efficiently if it interacts with the mature part of the precursor. As Mas37p/Mas70p preferentially recognizes precursors that are bound to ATP-requiring cytosolic chaperones, it may also interact with these chaperones. A promising partner for such an interaction is the mitochondrial import stimulating factor MSF; MSF is an ATPase which prevents aggregation of mitochondrial precursors by binding them in an incompletely folded conformation(28) .
A complex containing at least three of
the four known receptor subunits shown in Fig. 8can be isolated
from digitonin-solubilized mitochondria (29) ; its existence in vivo is also suggested by the observation that the
cytosolic domains of Mas20p and Mas70p interact with each other in the
two hybrid system. This complex may ensure efficient and
specific binding of mitochondrial precursor proteins by simultaneously
recognizing different portions of a precursor molecule.