* Department of Biological Sciences, University at Albany, SUNY, Albany, New York 12222; Department of Cell Biology,
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and § Department of Molecular Medicine,
Wadsworth Center, NYS Department of Health, Empire State Plaza, Albany, New York 12201-0509
We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals. To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane. We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity. We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation. In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides. Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane. Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.
IN eukaryotic cells, a key step in the sorting of proteins
to intracellular compartments is the translocation of
polypeptides across organelle membranes. Although
the mechanisms of these processes are not well understood,
it has been suggested that proteins may cross membranes
through pores or channels (Blobel and Dobberstein, 1975 Most mitochondrial proteins are synthesized in the cytosol as precursor proteins, carrying amino-terminal extensions called presequences. Presequences carry the information that targets proteins to the mitochondrion and are
removed during or after import into the organelle. Precursor proteins are imported via a multi-step process that includes binding to outer membrane receptors, and translocation across one or both mitochondrial membranes. Translocation of the precursor across the mitochondrial
inner membrane requires an electrochemical potential
which is set up by the electron transport chain (Gasser et al.,
1982 Tim23 is an integral protein of the inner membrane essential for import (Emtage and Jensen, 1993 Both mitochondrial membranes contain a number of
channel activities which have been identified using electrophysiological techniques (Kinnally et al., 1992 We have recently shown that the conductance of MCC
is transiently blocked by synthetic peptides corresponding
to mitochondrial presequences (Lohret and Kinnally, 1995b Isolation of Mitochondria and Preparation
of Proteoliposomes
Mitochondria were isolated from wild-type strain AH216 and the tim23-1
mutant as described (Emtage and Jensen, 1993
Patch Clamp Analysis
The patch-clamp procedures and analysis used were as previously described (Lohret and Kinnally, 1995a Antibody Inhibition
Preimmune IgG and IgG against Tim23p were prepared from serum as
described (Emtage and Jensen, 1993 Peptides
Peptides were prepared by the Wadsworth Center's peptide synthesis core
facility using a 431A automated peptide synthesizer as previously described (Applied Biosystems, Foster City, CA) (Lohret and Kinnally,
1995b Table I.
The Effects of Synthetic Peptides on MCC Activity
).
Recently, Simon and Blobel (Simon and Blobel, 1991
, Simon and Blobel, 1992
) used electrophysiological techniques to identify potential protein-translocating channels
in the endoplasmic reticulum and in the bacterial plasma
membrane. In mitochondria, proteins imported from the
cytosol utilize import complexes in both the inner and
outer membranes (Pfanner et al., 1994
; Ryan and Jensen, 1995
; Pfanner and Meijer, 1995
; Lithgow et al., 1995
). However, the mechanism by which imported proteins cross either mitochondrial membrane is unclear.
; Schleyer et al., 1982
). In addition, a matrix-localized
member of the hsp70 family (mt-hsp70) plays an important role in the translocation of precursors across the inner
membrane (Kang et al., 1990
; Ungermann et al., 1994
, 1996).
The Tim44 protein is associated with the matrix face of the
inner membrane (Blom et al., 1993
; Horst et al., 1993
;
Maarse et al., 1992
; Scherer et al., 1992
) and interacts with
mt-hsp70 during the translocation reaction (Kronidou et
al., 1994
; Rassow et al., 1994
; Schneider et al., 1994
).
; Dekker et
al., 1993
). Mitochondria isolated from yeast strains carrying the tim23-1 mutation are defective in the import of at
least five different precursor proteins (Emtage and Jensen,
1993
). Furthermore, antibodies to Tim23p inhibit import
across the inner membrane (Emtage and Jensen, 1993
).
Tim23p can be chemically cross-linked to a precursor arrested in transit across the inner membrane (Ryan and Jensen, 1993
; Kübrich et al., 1994
), and depletion of Tim23p
from cells results in a defect in import (Emtage and Jensen, 1993
). Tim17p is another essential inner membrane
import component (Maarse et al., 1994
; Ryan et al., 1994
)
that associates with Tim23p (Blom et al., 1995; Berthold et al., 1996; Ryan, K.R., R. Leung, and R.E. Jensen, manuscript submitted for publication). While the exact function
of Tim23p and Tim17p in import is not known, it has been
suggested that both proteins form part of a channel in the
inner membrane through which precursors are translocated into the matrix.
; Sorgato and
Moran, 1993
). The multiple conductance channel (MCC1
or mitochondrial megachannel; Kinnally et al., 1996
; Zoratti and Szabó, 1994
) is a channel activity found in the mitochondrial inner membrane of mammals and yeast. MCC
has a large conductance and allows the passage of a variety
of different ions across the membrane in patch-clamp
studies (Lohret and Kinnally, 1995a
). This channel is normally closed under metabolizing conditions unless activated (Kinnally et al., 1991
, 1992
, 1996). However, MCC
activity is usually detected after its reconstitution into proteoliposomes, suggesting that regulatory components may
be lost during the fractionation procedure (Lohret et al.,
1996
).
).
Presequence peptides caused a momentary closure of MCC
(a flicker blockade) that is reversible, voltage-, and dosedependent. To determine whether MCC plays a role in mitochondrial protein import, we have examined the relationship between MCC and Tim23p, an inner membrane
import component. Below we find that antibodies to
Tim23p inhibit MCC activity. We also find the peptide sensitivity of MCC is altered in mitochondria isolated from
the tim23-1 mutant. Our results indicate that Tim23p is required for normal MCC activity, and suggest that precursors are translocated across the inner membrane through
the pore of the MCC.
Materials and Methods
; Daum et al., 1982
). Mitochondrial membranes were prepared by the French press method (Decker
and Greenawalt, 1977
), and the outer membrane was separated from the
inner membrane as described by Mannella (1982)
. The purity of the membrane fractions was assayed in two ways. First, immunoblotting showed,
for the most part, that the outer membrane protein, voltage-dependent
anion-selective channel (VDAC), was found only in the outer membrane
preparations, and that the inner membrane protein Tim23 was found
solely in the inner membrane preparation (see Fig. 1). Second, we found
that VDAC activity detected by patch-clamp analysis was found only in
outer membrane, but not in inner membrane preparations (Lohret and
Kinnally, 1995a
; Lohret et al., 1996
). Inner and outer membranes were
separately reconstituted into giant proteoliposomes (Sigma Type IV-S
soybean l-
-phosphatidylcholine) by dehydration-rehydration (Criado and
Keller, 1987
) as previously described (Lohret and Kinnally, 1995a
,b; Lohret
et al., 1996
). To eliminate the contribution of VDAC to the channel activity of outer membrane preparations, strain M22-2 (Blachly-Dyson et al.,
1990
), which is disrupted for VDAC, was used as the source of outer
membranes in studies of the Tim23 antibody on PSC (Lohret and Kinnally, 1995a
).
Fig. 1.
Analysis of mitochondrial inner and outer membrane
preparations. Immune blots indicate the presence of Tim23 in the
inner and VDAC in the outer membrane preparations. Aliquots
from inner (IM) and outer (OM) membrane preparations from
wild-type mitochondria were subjected to SDS-PAGE and immune blots were decorated with antibodies to VDAC (A) and
Tim23 (B) proteins. Immune complexes were visualized using
AuroProbe BLplus secondary antibody reaction.
[View Larger Version of this Image (23K GIF file)]
,b; Lohret et al., 1996
). Briefly, membrane patches were excised from proteoliposomes after formation of a
giga-seal using micropipettes with ~0.4 µm diameter tips and resistances
of 10-20 M
(program courtesy of A.K. Dean, Sutter Instruments, Novato, CA). Unless otherwise stated, the solution in the micropipettes and
bath was 150 mM KCl, 5 mM Hepes, 1 mM EGTA, 1.05 mM CaCl2
(10
5M free calcium), pH 7.4, and the experiments were carried out at
room temperature (~23°C). Peptides were either introduced and removed
by perfusion of the bath (0.5 mL vol) with 3-5 mL of media or included in
the micropipette filling solution. Voltage clamp was performed with the
inside-out excised configuration of the patch-clamp technique (Hamill et
al., 1981
) using a Dagan 3900 patch clamp amplifier in the inside-out
mode. Voltages across patches excised from proteoliposomes are reported
as bath (i.e., matrix) potentials. Channel open probability was calculated
as the fraction of the total time the channel spent in the fully open state
from total amplitude histograms generated with PAT program (Strathclyde Electrophysiological Software, courtesy of J. Dempster, U. of
Strathclyde, UK). Mean open times were determined from current traces
usually 20-40 s in duration, band-width limited to 2 kHz by a low pass filter, and sampled at 5 kHz using the PAT program. Flicker rate was the
number of transition events/sec from the open state to lower conductance
states with a 50% threshold of the predominant event (typically ~500 pS).
In comparisons of tim23-1 and wild-type MCC, the percent reduction in
flicker rate was [1
(N from tim23-1/N from wild-type)]100, where N was
the increase in flicker rate induced by the addition of the presequence
peptide [N = (rate with peptide)
(rate without peptide)]. Only single
channel patches were used for the analysis of flicker blockade. Permeability ratios were calculated from the reversal potential in the presence of a
150:30 mM KCl gradient as previously described (Lohret and Kinnally,
1995a
).
). To examine the effect of antibodies
on the intermembrane space face of the inner membrane, preimmune IgG
and IgG against Tim23p, VDAC (Stanley et al., 1995
), and the Rieske iron-
sulfur protein (Beckmann et al., 1987
) were added to proteoliposomes (25 µg antibody/µg inner membrane protein) and incubated on ice for 60 min
before the patch-clamp experiments.
). As shown in Table I, the presequence peptides used were based
on amino acids 1-13 and 1-22 from the amino terminus of cytochrome oxidase subunit IV of S. cerevisiae (yCOX-IV1-13 and yCOX-IV1-22), amino
acids 3-22 of N. crassa subunit IV (fCOX-IV), and amino acids 1-20 from
subunit VI of S. cerevisiae (yCOX-VI). Control peptides were the amino and
carboxy termini and an internal segment of N. crassa VDAC (nVDAC,
cVDAC, and iVDAC, respectively; Guo et al., 1995
), the binding domain
of antithrombin III (pAT-III; Smith and Knauer, 1987
), and a synthetic
mitochondrial presequence, synB2 (Allison and Schatz, 1986
). Peptides
were subjected to mass spectroscopy to determine impurities and proper
composition.
Immune Blotting
Mitochondrial proteins were separated by SDS-PAGE (Laemmli, 1970)
and transferred to nitrocellulose or Immobilon filters (Towbin et al.,
1979
). Filters were decorated with antibodies and visualized with an AuroProbe BLplus secondary antibody reaction (Amersham, Amersham, UK)
or by chemiluminescence (ECL; Amersham).
Conductance of MCC Is Transiently Blocked by Presequence Peptides
We previously showed that peptides based on two mitochondrial presequences transiently block the conductance
of MCC (Lohret and Kinnally, 1995b). We have extended
this investigation by examining the effect of additional
peptides on MCC activity. Mitochondrial inner membranes
were isolated from wild-type yeast strains and fused with
small phosphatidylcholine liposomes to form large proteoliposomes. Membrane patches were excised from the proteoliposomes with a micropipette and the conductance was
examined in the presence or absence of peptides. Peptides
were either added to the bath solution, which represents
the mitochondrial matrix, or added to the micropipette
buffer, representing the intermembrane space.
As shown previously, MCC in the absence of peptide
had a predominant transition size of ~500 pS, a peak conductance of ~1 nS, a mean open time of ~25 ms at 20 mV,
and a cation selectivity (Lohret and Kinnally, 1995a; see
also Figs. 2 and 3). When a peptide based on the first 13 residues of the Saccharomyces cerevisiae cytochrome oxidase subunit IV presequence (yCOX-IV1-13) was added to
the bath solution, a transient blockade of MCC conductance was induced during perfusion of the chamber as indicated by the decrease in mean current (Fig. 2 A). While
transitions to lower conductance levels were relatively infrequent in the absence of presequence peptide (seen as
downward deflections in the current trace of Fig. 2 B),
large amplitude, rapid flickering between the open and
lower conductance states developed in the current trace
during the introduction of yCOX-IV1-13 (Fig. 2 C) and persisted after the perfusion was complete (Fig. 2 D).
The reduced occupancy of the open state of MCC and
rapid flickering between open, sub-, and closed states
brought on by presequence peptides is further illustrated
by comparing the current amplitude diagrams and single
channel current traces in the absence (control) and presence of yCOX-IV1-13 in Fig. 3. The peptide-induced reduction in open probability from 0.9 (control) to 0.4 (yCOXIV1-13) was seen as a decrease in the fraction of the total
time spent in the open state peak of the amplitude diagrams. The transient blockade of MCC conductance by
presequence peptide was seen from either side of the
membrane, but the effect was voltage-dependent. When the presequence peptide was added to the bath, the rapid
flickering was seen at bath positive potentials (negative in
the micropipette), but not at bath negative potentials
(Lohret and Kinnally, 1995b). When the peptide was included in the micropipette, the blockade of MCC conductance was seen only at bath negative potentials. Several
additional presequence peptides induced rapid flickering and reduced open probability of MCC, including peptides
based on the amino terminus of yeast cytochrome oxidase
subunit IV (yCOX-IV1-22) and subunit VI (yCOX-VI), as
well as subunit IV of cytochrome oxidase from N. crassa
(fCOX-IV), and are scored positive in Table I.
Functional mitochondrial presequences contain several
basic amino acids and are thought to fold into amphipathic
helices (Roise and Schatz, 1988
). To test the specificity
of the peptide inhibition of MCC conductance, we examined several peptides either previously shown or not predicted to act as mitochondrial presequences. For example,
synB2 is a synthetic peptide that does not function as a mitochondrial targeting signal (Allison and Schatz, 1986
). synB2 is cationic and predicted to be
-helical, but has low
amphipathicity. We find that synB2 has little or no effect on
MCC activity compared to mitochondrial presequences,
e.g., yCOX-IV1-13, as shown in Fig. 3. yCOX-IV1-13 induced
large-amplitude flickering and reduced the open probability of MCC while the current traces and amplitude diagrams in the presence of synB2 closely resembled the control (Fig. 3). Furthermore, we found that nVDAC, an
-helical
peptide with one negative charge (Gou et al., 1995), cVDAC,
an uncharged peptide that is not
-helical (Gou et al., 1995)
as well as iVDAC, a cationic peptide predicted to form a
-sheet (Mannella et al., 1996
), did not cause the flicker
blockade in our MCC assays (scored as negative in Table
I). Interestingly, pAT-III (another cationic
-helical peptide
with low targeting activity; Glaser and Cumsky, 1990
) had
little effect on MCC activity if included in the micropipette but induced a flicker blockade of MCC if applied to the
bath face.
Increasing the net positive charge and/or length of presequence peptides increased their ability to competitively
inhibit protein import (Cumsky and Glaser, 1990) while
similar changes in presequences increased the efficiency of
protein import (Isaya et al., 1988; Martin et al., 1991
). The
ability of presequence peptides to induce rapid flickering of
MCC was similarly sensitive, i.e., more positively charged
or longer peptides had lower effective concentrations. In
particular, 2 µM yCOX-IV1-22 or yCOX-VI (+5 net charge)
had about the same effect on MCC as 20 µM fCOX-IV or
yCOX-IV1-13 (+3 net charge, data not shown).
Antibodies to Tim23p Block MCC Activity
The specific blockade of MCC conductance by presequence peptides suggests that MCC plays a role in mitochondrial protein import. To further test this idea, we
asked if antibodies to the Tim23 protein affect the activity
of MCC. Tim23p is a component of the protein import machinery and has been proposed to be part of a protein-translocating channel in the inner membrane (Ryan and Jensen,
1993; Ryan et al., 1994
; Dekker et al., 1993
). For example, translocation of precursors across the inner membrane can
be inhibited by antibodies to Tim23p (Emtage and Jensen,
1993
). We preincubated proteoliposomes prepared with
mitochondrial inner membranes with antibodies to Tim23p
and then examined the conductance of patches excised
from the treated vesicles. As shown in the current traces of
Fig. 4 A, antibodies to Tim23p blocked MCC activity. When
the conductance of patches from proteoliposomes preincubated with Tim23 IgG was measured, MCC was virtually absent. Tim23 IgG blocked essentially all conductance through MCC (Fig. 4 A) and blocked the effect of
yCOX-IV1-13 peptide. In contrast, equivalent amounts of
preimmune IgG did not affect MCC activity (Fig. 4 A). To
quantify the effect of Tim23 antibodies, several patches
were taken from proteoliposomes treated with Tim23 IgG
and their conductance was measured. As controls, proteoliposomes were also incubated with IgG from preimmune
serum, antibodies to the outer membrane VDAC channel
(Stanley et al., 1995
), or IgG to the Rieske iron-sulfur protein of the inner membrane electron transport chain
(Beckmann et al., 1987
) (Fig. 4 B). In a total of 28 patches
from proteoliposomes treated with Tim23 IgG, no MCC
activity was observed in 23 of the patches, whereas five
patches had detectable MCC (Fig. 4 B). In all of the controls (90 total patches), MCC activity was found in normal
amounts (Fig. 4 B).
To test the specificity of the inhibition of MCC by Tim23 antibodies, we examined the activity of an outer membrane channel activity, PSC (Fig. 4 C). Proteoliposomes prepared using outer membranes were incubated with equivalent amounts of Tim23 IgG or antibody from preimmune serum. Neither IgG had any effect on the detection level of PSC, and activity similar to untreated proteoliposomes was found in the preparations preincubated with either preimmune or Tim23 IgG (Fig. 4 C). Thus, antibodies to Tim23p appear to specifically inhibit the inner membrane channel MCC, and further support the possibility that MCC plays a role in mitochondrial protein import.
Conductance of MCC Isolated from the tim23-1 Mutant Is Not Transiently Blocked by Presequence Peptides
To further test the connection between MCC and protein
import, we examined MCC activity in mitochondria isolated from the tim23-1 mutant. The tim23-1 mutation results in a substitution of aspartate for glycine at position
186 in Tim23p (Emtage, 1994). Mitochondria isolated from
tim23-1 mutants are defective in the import of several different precursor proteins, including subunit IV of yeast cytochrome oxidase (Emtage and Jensen, 1993
). We prepared mitochondrial inner membranes from either wildtype or the tim23-1 mutant, reconstituted the membranes
into proteoliposomes, and then measured the conductance
of patches excised from the vesicles.
We found that the electrical properties of MCC isolated
from wild-type and tim23-1 strains were virtually identical
(compare control current traces of Fig. 5 A with 5 B and
Fig. 6 A with 6 B). In particular, MCC from both strains
had the same peak conductance, predominant transition size,
mean open time, and cation selectivity. Furthermore, permeability ratios for K+/Cl were ~6 for MCC from wildtype and tim23-1 patches with a 150:30 mM KCl gradient.
Conductance through MCC from both strains was voltage
dependent, i.e., MCC is predominantly open at low (e.g., 20 mV) but not high potentials of either polarity. The V0
(voltage where the probability of opening and closing are
the same) at positive potentials was less than V0 at negative potentials for MCC from both wild-type and tim23-1
strains.
In the presence of presequence peptides, however, MCC
activity from wild-type and tim23-1 strains differed dramatically. The conductance of MCC isolated from wild-type
strains is transiently blocked by presequence peptides
(Fig. 5 A, see also Figs. 2 and 3, and Table I). The flicker
rate of MCC from the wild-type strain increased ~10-fold
upon addition of the yCOX-IV1-13 peptide to the bath solution at positive potentials (Fig. 5 A). Similarly, the
flicker rate of wild-type MCC was ~10-fold higher at negative potentials if yCOX-IV1-13 was included in the micropipette buffer (Fig. 6 A). The flicker rate of MCC isolated from wild-type cells increased with yCOX-IV1-13
concentration in the bath and saturated by ~25 µM (Fig.
7). This dose-dependent inhibition of MCC is similar to that
required for the inhibition of protein import by presequence peptides (Glaser and Cumsky, 1990). In marked
contrast, the activity of MCC isolated from the tim23-1
mutant was only slightly affected by the addition of
yCOX-IV1-13 peptide (Figs. 5 B and 6 B). Quantification showed that the tim23-1 mutation caused at least an 85%
reduction in the transient conductance blockade (or increased flicker rate) induced by yCOX-IV1-13 and fCOXIV in the bath (Fig. 5 C). Similar results were also found
with yet another presequence peptide yCOX-VI at 2 µM
concentrations (data not shown). Furthermore, we found that peptides added to the micropipette, instead of the
bath, failed to induce flickering of MCC isolated from the
tim23-1 mutant (Fig. 6). The flicker rate of wild-type
MCC, but not tim23-1 MCC, was ~10-fold higher at negative potentials when the yCOX-IV1-13 peptide was present
in the micropipette solution. At the same time, the lower
open probability for wild-type MCC that is associated with the peptide-induced rapid flickering was not seen with
MCC from the tim23-1 strain (Fig. 6).
tim23-1 mutants are temperature-sensitive for growth
and mitochondrial protein import, but mitochondria isolated from tim23-1 cells are defective in protein import in
vitro at all temperatures (Emtage and Jensen, 1993). We
have recently found that the Tim23-1 protein is rapidly degraded during mitochondrial isolation from tim23-1 strains
(Ryan, K.R., R. Leung, and R.E. Jensen, manuscript submitted for publication). Similarly, we find that proteoliposomes prepared from tim23-1 inner membranes contain
greatly reduced levels of the Tim23-1 protein (Fig. 8).
Equal amounts of proteoliposomes prepared from wildtype and tim23-1 mitochondrial inner membranes were analyzed by immune blotting. While similar levels of cytochrome oxidase subunit IV (Cox4p; Fig. 8) and the
-subunit of the F1-ATPase (not shown) were seen in the two
preparations, the level of the Tim23 protein was reduced
at least 10-fold in the tim23-1 proteoliposomes. Importantly, the frequency of detecting MCC in proteoliposomes prepared from similar quantities of wild-type and
tim23-1 inner membrane protein was virtually the same.
Twelve MCC were detected in thirteen patches from proteoliposomes prepared with 16 µg tim23-1 inner membrane
protein/mg lipid, while 0, 7, and 32 MCC were recorded from fifteen patches each from proteoliposomes prepared
with 3, 27, and 133 µg wild-type inner membrane protein/
mg lipid, respectively. Hence, the reduced level of Tim23
protein in the inner membrane of tim23-1 mitochondria
may explain their defect in protein import, as well as the
alteration (but not loss) of MCC activity.
tim23-1 Mutants Are Not Altered in the Activity of PSC, an Outer Membrane Peptide-sensitive Channel
To show that the tim23-1 mutation specifically affected
MCC activity, we examined the activity of the outer membrane peptide-sensitive channel, PSC. Like MCC in the inner membrane, the conductance of PSC is transiently
blocked by presequence peptides (Henry et al., 1996;
Fèvre et al., 1994
; Theiffry et al., 1992; Juin et al., 1995
).
Outer membranes were isolated from mitochondria of wild-type cells and the tim23-1 mutant, and then reconstituted into proteoliposomes. When membrane patches were
examined, we found that the PSC activities from both
preparations were comparable in terms of conductance,
selectivity, and voltage dependence. Furthermore, addition of the yCOX-IV1-13 peptide increased the flicker rate
of PSC from both wild-type and tim23-1 strains by ~10fold as shown by the current traces and histograms of Fig. 9.
This inhibition was both dose- and voltage-dependent
(data not shown). In addition, we previously found deletion of VDAC, or the adenine nucleotide translocator had
no effect on MCC activity in proteoliposomes (Lohret and
Kinnally, 1995a
; Lohret et al., 1996
). Our results indicate
the tim23-1 mutation specifically alters the activity of the
inner membrane MCC, and has no effect on the outer
membrane PSC activity. The Tim23 antibody and the tim23-1 mutation allow discrimination between MCC and
PSC activities.
Our studies indicate a striking correlation between MCC
activity and the translocation of precursors across the inner membrane. We find that Tim23p, an inner membrane
import component, is required for peptide-sensitive activity
of MCC. Antibodies to Tim23p block both protein import
and the MCC channel. We find that MCC conductance is
specifically blocked by presequence peptides. Furthermore, we find that the presequence peptide sensitivity of MCC
isolated from the protein import deficient mutant tim23-1
is dramatically reduced. MCC has several properties expected for a protein-translocating channel in the mitochondrial inner membrane. For example, the pore size of MCC
is estimated at 2-3 nm (Zoratti and Szabó, 1994), which is
sufficiently large to allow the passage of unfolded precursor proteins. In addition, the predominant transition size
of MCC (500 pS) is similar to potential protein-conducting
channels observed both in the endoplasmic reticulum and
the bacterial plasma membrane (Simon and Blobel, 1991
,
1992
).
Biochemical studies indicate that the membrane potential drives the initial movement of the presequence through
the inner membrane and that the presequence in transit is
not tightly bound to the import apparatus (Martin et al.,
1991; Berthold et al., 1995
; Ungermann et al., 1996
). Thus,
the inner membrane import channel is proposed to be a
passive pore interacting only weakly with translocating
polypeptide chains (Ungermann et al., 1994
). Consistent
with this idea, we found that the transient blockade of
wild-type MCC by presequence peptides could occur from
either side of the membrane in a potential-dependent
manner, i.e., at positive potentials (pipette negative) when
the presequence peptide was added to the bath and at negative potentials (pipette positive) when peptide was included in the pipette. In addition, flicker rate increased
with the magnitude of the voltage. Moreover, the reduction in open probability and increase in flickering induced by addition of presequence peptide to the bath was reversed by perfusion with fresh media, suggesting the presequence peptides are not tightly bound. The peptide-
induced flickering, however, was specific for presequence
peptides. For example, peptides shown not to function as
presequences, such as synB2 (Allison and Schatz, 1986
; Roise
et al., 1988), or peptides that were not cationic, did not affect MCC activity.
The presequence peptide-induced flickering between
open and subconductance states seen in wild-type MCC
may be due to the momentary occlusion of the channel
during translocation of the peptide from one side of the
membrane to the other. Although direct demonstration of
peptide translocation through MCC will likely require reconstitution of MCC using purified components, a similar peptide-induced flickering seen in studies with the outer
membrane channel, PSC, was shown to be associated with
the movement of peptide across the membrane (Thieffry
et al., 1992; Juin et al., 1995
; Henry et al., 1996
). Alternatively, the rapid flickering of MCC may result from a destabilization of the open state upon binding of the peptide
to one or more sites on MCC. Since MCC isolated from
the tim23-1 mutant is not blocked by presequence peptides
added to either side of the membrane, we suggest that the
tim23-1 mutation may either hinder translocation of presequence peptides through MCC or alter presequence peptide recognition by MCC.
Mitochondria must maintain an electrochemical gradient for ATP synthesis. Therefore, any protein-translocating channel in the inner membrane must be tightly regulated. While MCC is normally closed in mitochondria
under metabolizing conditions, it is usually detected after
inner membranes are reconstituted in liposomes. Presumably a regulatory component is lost during reconstitution that normally maintains MCC in a closed state in the absence of protein import. In preliminary studies, we find
that an inner membrane pore, presumably MCC, can be
opened in isolated mammalian mitochondria and mitoplasts under normal conditions by presequence peptides
(Sokolove and Kinnally, 1996; Campo, M.L., and K.W.
Kinnally, unpublished data).
The tim23-1 mutation results in a substantial reduction in
the import of several different precursors into isolated mitochondria (Emtage and Jensen, 1993). Although tim23-1
strains are temperature-sensitive for viability, the protein
import defect in vitro was seen at the permissive temperature. Consistent with previous results (Ryan, K.R., R. Leung,
and R.E. Jensen, manuscript submitted for publication), we found the Tim23 protein was reduced 10-20-fold in mitochondria isolated from the tim23-1 mutant as compared
to the wild-type strain. Associated with loss of Tim23 protein and a defect in protein import, we found that the activity of MCC from tim23-1 mitochondria was markedly
insensitive to presequence peptides. In the absence of peptide, however, MCC activity from wild-type strains and the
tim23-1 mutant was similar. For example, no significant differences in pore size (as reflected by peak conductance), predominant transition size, voltage dependence,
or ion selectivity were seen. Furthermore, the number of
channels detected in the wild-type and tim23-1 membrane
preparations was virtually the same. Our findings that the
Tim23 protein is reduced in tim23-1 mitochondria suggests
that Tim23p is not a structural component of the pore of
MCC. Instead, we argue that Tim23p plays a regulatory
function in MCC activity.
The Tim23 protein contains a 9-kD hydrophilic aminoterminal domain facing the intermembrane space, and four
potential carboxyl-terminal membrane-spanning segments
(Bauer et al., 1996; Emtage, J.L.T., and R.E. Jensen, manuscript submitted for publication). The amino terminus of
Tim23p appears to function as a receptor for presequences as they are translocated through the outer membrane import machinery (Emtage et al., 1993; Bauer et al., 1996
). In
addition, Tim23p has been shown to form dimers in mitochondria in a potential-dependent manner (Bauer et al.,
1996
). Dimerization of Tim23p is disrupted upon the binding of precursors. The role of Tim23, therefore, may be to
bind the presequence and pass the precursor to the inner membrane import channel. In this view, tim23-1 mutants
would be defective in the recognition of the presequence,
but MCC activity would not otherwise be affected. To further test the relationship between MCC and mitochondrial
protein import, we are currently analyzing MCC defective
in or lacking different import components.
Timothy A. Lohret's present address is Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 303223030.
Received for publication 6 June 1996 and in revised form 22 January 1997.
1. Abbreviations used in this paper: MCC, multiple conductance channel; PSC, peptide-sensitive channel; VDAC, voltage-dependent anion-selective channel.We thank Carmen Mannella, Robert Murphy, and Richard Zitomer for their helpful discussions and for reading this manuscript. We thank Andre Moodie, Roxanne Leung, and Ana Rodriguez for technical assistance. Antibody raised against N. crassa VDAC was graciously provided by C.A. Mannella (Wadsworth Center, NYS Dept. of Health [NYSDOH], Albany, NY). Strain M22-2 was graciously provided by Michael Forte (Oregon Health Science University, Portland, OR). We also thank J. Dias (Wadsworth Center, NYSDOH, Albany, NY) for the synthesis of peptides and B. Trumpower (Dartmouth) for antibodies against the iron sulfur protein of complex I.