(Received for publication, August 21, 1995; and in revised form, September 28, 1995)
From the
MAS17 (MAS22) is an essential component of the import receptor
complex in the yeast mitochondrial outer membrane. MAS17 consists of
three distinct domains: the N-terminal cytosolic domain, the internal
membrane-spanning domain, and the C-terminal intermembrane space
domain. In the present study, we examined the roles of the C-terminal
domain of MAS17, which is rich in acidic amino acids, in protein import
into mitochondria both in vivo and in vitro. Cells
expressing MAS17120-152, a mutant MAS17 lacking the
C-terminal acidic domain, could grow as fast as those expressing
wild-type MAS17, while cells expressing MAS17
97-152, a
mutant MAS17 lacking both the intermembrane space and the
membrane-spanning domains, stopped growing as soon as wild-type MAS17
was depleted. MAS17
120-152 was correctly integrated into the
mitochondrial outer membrane like wild-type MAS17. Mitochondria
containing MAS17
120-152 instead of wild-type MAS17 could
import both authentic and artificial mitochondrial precursor proteins
nearly as efficiently as wild-type mitochondria in vitro.
These results suggest that the C-terminal intermembrane space domain of
MAS17 is not essential for targeting or functions of MAS17.
Nuclear-encoded mitochondrial precursor proteins are imported from the cytosol into mitochondria; they are recognized by cytosolic factors and/or mitochondrial receptor proteins and are subsequently translocated across the outer and inner mitochondrial membranes(1, 2) . Translocation across the mitochondrial membranes requires coordinated functions of two separate machineries in the outer and inner mitochondrial membranes; they most likely interact transiently to allow the movement of precursor proteins into the matrix(3, 4) .
MAS20 (5) and MAS70 (6) in Saccharomyces cerevisiae and MOM19 (7) and MOM72 (8) in Neurospora crassa serve as receptor proteins and are responsible for the initial binding of mitochondrial precursor proteins to the mitochondrial surface. Yeast MAS37 may form a heterodimer complex with MAS70 and facilitate the receptor function of MAS70(9) . ISP42 in yeast (10) and MOM38 in N. crassa(11) likely mediate the step of subsequent protein translocation across the outer membrane. ISP42/MOM38 was found to be in contact with precursor proteins arrested in transit across the mitochondrial membranes(12, 13) , and antibodies against ISP42 blocked protein import into mitochondria(12) .
Another outer membrane protein MOM22 in N. crassa appears to function downstream of the receptors because antibodies against MOM22 inhibited import of mitochondrial precursor proteins into mitochondria but not their binding to receptors in vitro(14) . MAS17 (MAS22), the yeast equivalent of N. crassa MOM22, has been identified recently(15, 16, 17) . The MAS17 gene is essential (15, 16, 17) and depletion of functional MAS17 results in accumulation of the precursor form of a mitochondrial protein(16) . MAS17 consists of three distinct domains: the N-terminal domain that is highly acidic and faces the cytosol, the internal hydrophobic domain that is integrated into the outer membrane, and the C-terminal domain that contains several acidic residues and faces the intermembrane space. The cytosolic acidic domain may well provide a binding site for positively charged presequences of mitochondrial precursor proteins.
A key question
concerning the protein import into mitochondria is what drives the
vectorial movement of precursor proteins across the two membranes. In
the case of protein translocation across the inner mitochondrial
membrane, the membrane potential across the inner membrane triggers the
movement of a positively charged presequence across the inner membrane
probably by electrophoretic effects(18, 19) . Then
mitochondrial hsp70 ()likely drives the movement of the rest
of the precursor polypeptide chain at the expense of ATP hydrolysis in
the matrix(20, 21, 22) .
On the other hand, mechanisms of protein translocation across the mitochondrial outer membrane are poorly understood. Translocation of many matrix-targeting precursor proteins, which do not fold in the intermembrane space, across the outer membrane can be likely driven by their passage across the inner membrane. However, at least for the initiation of the translocation process, precursor proteins have to move across the outer membrane by using only the outer membrane machinery until their presequences can interact with the inner membrane machinery. Then, what drives the initial translocation of the mitochondrial proteins across the outer membrane? It has been recently shown that isolated mitochondrial outer membrane vesicles without the inner membrane have the ability to translocate the presequence, but not the mature part, of precursor proteins(23, 24) . The translocation of the presequence of the precursor proteins into the outer membrane vesicles involves at least two sites of presequence recognition at the outer membrane, one on the cis side and the other on the trans side (24) . Binding of the presequence to the ``trans'' site may promote unfolding of the precursor protein and translocation of the presequence across the outer membrane. An interesting hypothesis is that the acidic domain of MAS17 in the intermembrane space plays the role of the trans site; binding of the intermembrane space domain of MAS17 to the positively charged presequences of mitochondrial precursor proteins may pull precursors out of the import channel and into the mitochondria(15) .
In the present study, we examined the roles of the C-terminal acidic domain of MAS17 in protein import into mitochondria both in vivo and in vitro. The mutant MAS17 lacking the C-terminal acidic domain could mediate protein import into mitochondria as efficiently as wild-type MAS17. This suggests that the C-terminal domain of MAS17 is not essential for targeting and functions of MAS17.
Figure 1:
C-terminal regions of wild-type MAS17,
MAS17120-152, and MAS17
97-152. The C-terminal
regions of wild-type and mutant MAS17 used in this study are shown with
the single letter code. Acidic and basic amino acid residues are marked
with - and +, respectively. The boxes indicate the
putative membrane-spanning domains.
Since MAS17 is an essential gene of
yeast(15, 16, 17) , we first constructed a
strain that expresses conditionally the MAS17 gene from the
galactose-inducible GAL1 promoter. This strain, MNMS-1C,
harbors a chromosomal disruption of the MAS17 gene, but is
rescued by a single copy of MAS17 on a CEN4-ARS1-based plasmid (pYE-Ura3:MAS17). The pYE-Ura3:MAS17
plasmid contains the coding region of MAS17 fused to the yeast GAL1 promoter. The MNMS-1C strain could grow on galactose
containing medium but not on medium supplemented with glucose (not
shown). The two mutant mas17 genes, mas17120-152 and mas17
97-152,
as well as wild-type MAS17 and the vector alone, were
separately transformed into the MNMS-1C cells carrying pYE-Ura3:MAS17,
and the transformants were first selected on galactose-containing
medium, where wild-type MAS17 was expressed from pYE-Ura3:MAS17. As
shown in Fig. 2A, all the transformants could grow
normally on galactose-containing medium. The transformants were then
streaked onto glucose-containing medium where the expression of
wild-type MAS17 was repressed, and abilities of the co-transformed
mutant mas17 genes to complement the depletion of wild-type
MAS17 were analyzed. Although cells bearing the mas17
97-152 gene could not grow on
glucose-containing medium (Fig. 2B, sector 3), those
carrying the mas17
120-152 gene could grow on
glucose-containing medium as fast as those carrying the wild-type MAS17 gene (Fig. 2B, sectors 2 and 4). These results indicate that the membrane-spanning domain,
but not the intermembrane space domain is essential for the function of
MAS17 in vivo.
Figure 2:
Growth of yeast transformants harboring
the wild-type or mutant MAS17 gene. A and B,
the MNMS-1C cells carrying vector pRS314 alone (sector 1),
pRS314:MAS17 (sector 2), pRS314:MAS1797-152 (sector 3), or pRS314:MAS17
120-152 (sector
4) were streaked onto 2% galactose-containing medium (A),
or 2% glucose-containing medium (B), and were incubated for 3
days at 30 °C. C, the MNMS-MAS17 and the
MNMS-MAS17
120-152 cells (see text) were inoculated in 2%
glucose-containing or 3% glycerol containing medium, and cell growth in
each culture was followed by monitoring the absorbance at 600 nm. All
media contained 1% yeast extract and 2%
polypeptone.
To analyze the ability of mas17120-152 to complement the MAS17 deficiency
more precisely, we isolated yeast cells that retained either the
wild-type MAS17 gene (MNMS-MAS17) or the mas17
120-152 gene (MNMS-MAS17
120-152) on
a pRS314 plasmid but had lost the pYE-Ura3:MAS17 plasmid. We then
compared growth rates of the strains, MNMS-MAS17 and
MNMS-MAS17
120-152 (Fig. 2C). The cells
expressing MAS17
120-152 could grow nearly at the same rate
as those with wild-type MAS17 under fermentable conditions (Fig. 2C (glucose)) and at a slightly lower rate than
those with wild-type MAS17 under nonfermentable conditions (Fig. 2C (glycerol)). This means that
MAS17
120-152 is almost fully functional in vivo.
In order to compare the
topology of MAS17120-152 in mitochondria with that of
wild-type MAS17, we analyzed the sensitivity of MAS17
120-152
and MAS17 in intact mitochondria to trypsin (Fig. 3). As shown
in Fig. 3A (a), treatment with 40 µg/ml of
trypsin led to partial degradation of wild-type MAS17 to a fragment
with an apparent size of 12 kDa (lane 2, asterisk). The
fragment was hardly degraded even by 160 µg/ml of trypsin (lane
4) as long as intactness of the mitochondrial outer membrane was
retained (Fig. 3A (b)). However, the 12-kDa
fragment was degraded at lower concentrations of trypsin upon exposure
of the intermembrane space by hypotonic swelling (Fig. 3A (a), lanes 6-8), excluding the possibility
that it is intrinsically protease resistant. This 12-kDa fragment
likely contains the C-terminal intermembrane space domain as shown for N. crassa MOM22 previously(14) . Treatment with 40
µg/ml of trypsin also partially degraded MAS17
120-152 in
intact mitochondria (Fig. 3B (a), lanes
1-4), suggesting that, like MAS17, MAS17
120-152
without the C-terminal domain is integrated into the outer membrane
with its N-terminal domain exposed to the cytosol. As expected, in this
case, trypsin treatment did not lead to the formation of the 12-kDa
fragment (Fig. 3B (a), lanes
1-4).
Figure 3:
MAS17120-152 and wild-type
MAS17 are accessible to trypsin in intact mitochondria. Mitochondria
and mitoplasts were prepared from the MNMS-MAS17 (A) and
MNMS-MAS17
120-152 (B) strains and were treated with
indicated concentrations of trypsin for 30 min on ice. After the
proteolysis was stopped by addition of phenylmethylsulfonyl fluoride,
mitochondria (lanes 1-4) and mitoplasts (lanes
5-8) were recovered by centrifugation. Proteins in each
fraction were analyzed by SDS-PAGE and immunoblotting with anti-MAS17 (a), anti-Ssc1p and anti-cytochrome b
antibodies (b). Protection of cytochrome b
and Ssc1p against trypsin digestion reflect
intactness of the mitochondrial outer membrane and of the outer and
inner membranes, respectively. The asterisk indicates the
12-kDa fragment (see text).
Figure 4:
Solubilization of MAS17120-152
and wild-type MAS17 with urea and with digitonin from mitochondria. A, mitochondria (1 mg of protein) containing wild-type MAS17
or MAS17
120-152 were suspended in 100 µl of 20 mM HEPES-KOH, pH 7.4, with or without 4 M urea. After
incubation for 30 min on ice, mitochondrial membranes were recovered by
centrifugation at 20,0000
g for 10 min. Proteins in
the resulting pellet (P) and the supernatant (S) were
analyzed by SDS-PAGE and immunoblotting with the anti-MAS17 antiserum.
The total amounts recovered in both fractions (P + S)
were taken as 100%. B, mitochondria containing wild-type MAS17 (a) or MAS17
120-152 (b) were treated with
various concentrations of digitonin as described under
``Experimental Procedures.'' Proteins solubilized with
indicated concentrations of digitonin were analyzed by SDS-PAGE and
immunoblotting with the antibodies against MAS17, MAS20, ISP42, and
MAS70. The amount of each protein in untreated mitochondria was taken
as 100%.
We next solubilized mitochondrial
membrane proteins with increasing concentrations of a detergent,
digitonin (Fig. 4B). MAS17120-152 was
extracted with nearly the same concentration of digitonin as wild-type
MAS17, suggesting that MAS17
120-152 is anchored to the
mitochondrial outer membrane as tightly as wild-type MAS17.
Interestingly, concentration dependencies of the solubilization of
MAS17 and MAS17
120-152 parallel with ISP42 and MAS20,
whereas MAS70 was extracted at lower concentrations of digitonin than
MAS17, MAS17
120-152, ISP42, and MAS20. This is consistent
with the model that MAS17 forms a core complex of the protein
translocation machinery in the outer membrane with ISP42 and MAS20 but
not with MAS70. (
)
Figure 5:
MAS17120-152-containing
mitochondria import precursor proteins as efficiently as wild-type
mitochondria in vitro. Kinetics of in vitro import of
precursor proteins were compared between mitochondria prepared from the
MNMS-MAS17 strain (open circles) and those from the
MNMS-MAS17
120-152 strain (closed circles) as
described under ``Experimental Procedures.'' Import of
pF
(A) and pCOXIV-DHFR (B) were
performed at 30 °C, whereas import of pSu9-DHFR (C) and
urea-denatured pCOXIV-DHFR (D) were at 16 °C. Quantitative
analyses were carried out after separation of mitochondrial proteins by
SDS-PAGE and fluorography, followed by densitometry scanning of the
mature forms.
In the present study, we examined the role of the acidic
C-terminal domain of MAS17 in protein translocation across the
mitochondrial outer membrane both in vivo and in
vitro. The strain containing mutant MAS17
(MAS17120-152) that lacks the intermembrane space domain
grew as fast as the wild-type strain under fermentable or
nonfermentable conditions. MAS17
120-152 was integrated into
the mitochondrial outer membrane with the same topology as wild-type
MAS17. Mitochondria containing MAS17
120-152 could import
several mitochondrial precursor proteins as efficiently as those
containing wild-type MAS17 in vitro. A possible defect of
MAS17
120-152 may have been perhaps suppressed by increase in
the amount(s) of other component(s) of mitochondrial protein
translocation machineries. However, this is probably not the case,
since replacement of MAS17 by MAS17
120-152 in mitochondria
did not significantly affect the amounts of such proteins as MAS20 and
MAS70, receptor proteins in the outer membrane, ISP42, the component of
the translocation machinery in the outer membrane, MIM17, MIM23, and
MIM44, the components of the translocation machinery in the inner
membrane (32) , and Ssc1p, mitochondrial hsp70 in the matrix
that mediates protein translocation across the inner membrane (not
shown). Taken together, we conclude that the C-terminal domain of MAS17
is not essential for its role in protein import into mitochondria. This
means that, although MAS17 may facilitate protein translocation across
the outer membrane by binding to precursor proteins on the cytosolic
side of the membrane, it does not pull the precursor protein from the
trans side of the outer membrane.
Targeting signals of mitochondrial outer membrane proteins that have a single transmembrane segment near the N terminus or the C terminus were studied previously. In the case of MAS70 with a transmembrane segment near the N terminus, the positively charged, extreme N-terminal region functions as a targeting sequence, whereas the subsequent transmembrane segment is necessary for stop-transfer and anchoring functions(33) . The transmembrane segment of MAS70 alone is also capable of targeting and inserting a passenger protein(34) . In the case of the Bcl-2 protein with a transmembrane segment near the C terminus, the C-terminal region, including the transmembrane segment function as a signal-anchor sequence selective for the mitochondrial outer membrane, whereas association with the endoplasmic reticulum and nuclear envelope occurs by a different mechanism(35, 36) . MAS17 is unique in that it has a transmembrane segment in the middle of the polypeptide chain. The present study shows that correct targeting of MAS17 to the mitochondria was not impaired by deletion of the C-terminal intermembrane space domain, suggesting that it does not encompass the targeting signal of MAS17 for the mitochondrial outer membrane. Since in vitro import of N. crassa MOM22 into isolated mitochondria strictly depends on the surface receptor, MOM72 and MOM19(37) , identification of the targeting signal of MAS17 should be important for understanding the substrate specificity in the recognition of mitochondrial outer membrane proteins by the mitochondrial protein receptors.