From the
The effects of synthetic targeting peptides on the activity of
the multiple conductance channel (MCC) of mouse and yeast mitochondria
were investigated using patch-clamp techniques. Amino-terminal
targeting peptides of two inner membrane proteins reversibly decreased
the open probability and mean open time of MCC. One of these targeting
peptides had no effect on two other voltage-dependent mitochondrial
channels. Furthermore, the effects induced by the two targeting
peptides on MCC were not elicited by two peptides of an outer membrane
protein. The specific interactions of targeting peptides with MCC
suggest that this channel may be involved in protein import across the
inner mitochondrial membrane.
Since mitochondria are surrounded by two membranes, precursor
proteins targeted to the matrix must cross both barriers, presumably
through proteinaceous channels(1, 2, 3) .
Several membrane proteins participate in this process of translocation,
either through recognition of the precursors or their selective
passage. Components of the inner membrane import channel in yeast
mitochondria include MIM17 and MIM23 (e.g. see Refs. 2 and
4-6). A ``general insertion pore'' has been identified
in the outer membrane import apparatus, which contains proteins MOM38
and ISP6 and probably MOM30, MOM8, and MOM7(2) . While the inner
and outer membrane import machinery can operate independently, current
models favor their transient linkage at contact sites (junctions where
the two membranes are closely apposed)(2) . There is
considerable evidence that targeting sequences at the amino termini of
precursor proteins fold as cationic amphiphilic
Several channel activities, which
might function in the import of proteins into mitochondria, have been
identified in both the inner and outer mitochondrial membranes using
electrophysiological techniques (8-13). One of the channels has a
peak conductance state of 1-1.5 nS
To determine a possible link between MCC and
protein import, the effects of mitochondrial targeting peptides on this
channel activity have been studied and are described in this
communication.
Targeting peptides have been used
extensively to study protein import. For example, a yeast COX-IV
peptide blocks import of several mitochondrial proteins, probably at
the translocation
step(24, 32, 33, 34, 35) .
Several import-apparatus proteins, including Mas70p, ISP42, and a
28-kDa protein, have been identified by their ability to be
cross-linked to targeting peptides, e.g. yeast COX-IV
peptides(24, 34) .
However, the nature of the
interaction between the targeting peptides and MCC is not known. The
targeting sequences of most mitochondrial inner membrane proteins are
cationic and form amphiphilic
We thank Garfield Batchelor and Robert Murphy for
their technical support and Drs. Carmen Mannella, Henry Tedeschi, and
Richard Zitomer for discussions and review of this manuscript. We thank
the Wadsworth Center Biochemistry Core for the peptides.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helices and are
involved in both recognition and translocation of these
proteins(2, 7) .
(
)and
exhibits multiple conductance states, with transitions of 300-600
pS (0.15 M KCl) predominating. This multiple conductance-level
channel (MCC) is calcium-activated, slightly cation-selective,
and voltage-dependent, occupying low conductance levels at small
positive potentials(8, 10, 11) . The maximum
pore diameter of MCC is estimated at 2.7 nm (assuming a single barrel
pore)(13) . This channel activity is detected using patch-clamp
techniques in both mammalian (9, 10, 12) and
yeast mitoplasts (11) (mitochondria with the outer membrane
partially removed) as well as in purified inner membranes reconstituted
as proteoliposomes(11) . Although this activity was initially
described in 1989(8) , no definitive function or protein(s) have
been ascribed to MCC.
Mitochondria, Mitoplast, and Proteoliposome
Preparations
Mitochondria were isolated from mouse kidney as
described previously (8) and used to prepare mitoplasts
(3-5-µm diameter) by the French press method of Decker and
Greenawalt (14) to remove outer membranes. Mitochondria were
isolated from Saccharomyces cerevisiae by a modification of
the method of Daum et al.(15) . Outer mitochondrial
membranes were purified using the method of Mannella (16) with
the pellet from the last gradient spin used as the inner membrane
preparation. Yeast MCC was studied after reconstitution of inner
membranes because of the difficulty of patching the very small
(0.3-1.5 µm) diameter yeast mitoplasts(11) . Inner and
outer membranes were reconstituted into giant liposomes (Sigma type
IV-S soybean L--phosphatidylcholine) using the modified
dehydration-rehydration method of Criado and Keller (17) as
described previously(11, 18, 19) . Two yeast
strains were used for this study: wild type (M-3) and a strain
(M22-2) lacking the outer membrane voltage-dependent
anion-selective channel (VDAC)(20) . Both strains were
generously provided by M. Forte (Oregon Health Science University).
Patch Clamping
The patch-clamp procedures and
analysis used were essentially the same as described
previously(18, 19) . Membrane patches were excised from
mitoplasts and proteoliposomes after formation of a giga-seal using
micropipettes with 0.4-µm diameter tips and resistances of
20 megaohms. Unless otherwise stated, patching solution in the
pipette and bath was 0.15 M KCl, 5 mM HEPES, 1 mM EGTA, 1.05 mM CaCl
(10
M free calcium), pH 7.4. Peptides were introduced and
removed by perfusion of the bath (0.5-ml volume) with 3-5 ml of
media. Clamping voltage is reported as V = V
- V
, which
is relative to the mitochondrial matrix in inside-out excised mitoplast
patches. Channel open probability was calculated as the fraction of the
total time the channel spent in the fully open state from amplitude
histograms generated with the PAT program (Strathclyde
Electrophysiological Software, courtesy of J. Dempster, University of
Strathclyde, UK). Mean open times were determined from current traces
usually 20-40 s in duration. Current traces were bandwidth
limited to 2 kHz and sampled at 5 kHz using the PAT program.
Peptides
Peptides were prepared by the Wadsworth
Center's peptide synthesis core facility using an Applied
Biosystems 431A automated peptide synthesizer. The targeting peptides
used were the amino termini of cytochrome oxidase subunit IV (COX-IV)
and subunit VI (COX-VI). The control peptides were the amino and
carboxyl termini of VDAC, an outer membrane channel protein. Peptides
were subjected to mass spectroscopy to determine impurities and proper
composition. Circular dichroism spectra of COX-VI were taken on a Jasco
720 spectropolarimeter in trifluoroethanol to determine -helical
content (21).
MCC Is Specifically and Reversibly Affected by
Mitochondrial Targeting Peptides
The possible involvement of the
MCC in mitochondrial protein import was explored by determining the
effects of targeting peptides on channel behavior using patch-clamp
techniques. Targeting peptides induced decreases in the open
probability and mean open time of mouse and yeast MCC within seconds of
their introduction to the bath by perfusion. As shown in Fig. 1,
the targeting peptide from subunit IV of cytochrome oxidase (COX-IV)
induced a voltage-dependent change in wild-type yeast MCC behavior (n = 6 patches). The occupation of the fully open state
decreased while that of the major half-open substate and fully closed
state increased in the presence of COX-IV peptide at positive
potentials. Similar results were obtained with the targeting peptide
from subunit VI of cytochrome oxidase (COX-VI) (n = 6
patches). The peptide-induced effects were reversed by washing with
media (no peptide) as shown in Fig. 2(n = 7
patches). The same reversible effects were induced by COX-IV on MCC
activity from mouse (n = 2 patches) and yeast lacking
VDAC (n = 8 patches). The yeast strain lacking VDAC was
used because of similarities of VDAC and MCC. (The difference in MCC
voltage dependence noted between the two strains of yeast, M3 and
M22-2, when recording from mitoplasts (11) was lost with
reconstitution.(
))
Figure 1:
Wild-type yeast MCC activity is
affected by a targeting peptide. Current traces of MCC recorded from an
excised patch from a proteoliposome enriched in inner membranes at
various voltages are shown in the presence and absence of 50 µM COX-IV peptide. O, S, and C correspond
to the open, substate, and closed conductance
levels.
Figure 2:
The
effect of targeting peptides is reversible. Current traces (A)
and current amplitude histograms (B) (from current traces
20-30 s in duration; bin width, 0.4 pA) of wild-type yeast MCC at
30 mV are shown in the presence (middle) and absence (left) of 50 µM COX-IV peptide. Effects are
reversed by replacing the bath with media containing no peptide (right). Zero current level is measured at 0 mV under
symmetrical conditions. A slight loss of seal from 8 to 4 gigaohms
is observed after washing in this patch. Other conditions and
abbreviations are as described in the legend to Fig.
1.
The changes induced by
inner membrane targeting peptides in MCC were not observed with
synthetic peptides from either the carboxyl or amino terminus of the
outer membrane protein VDAC (n = 4 yeast patches for
each peptide at 50-100 µM, data not shown). The
blockade by targeting peptides and lack of effect by the VDAC peptides
could be observed with the same patch.
Targeting Peptides Affect MCC but Not Two Other
Mitochondrial Channel Activities
The peptide sensitivity of two
other mitochondrial channel activities was examined as a further test
of specificity. The presence of COX-IV targeting peptide caused no
obvious changes in the electrophysiological behavior of the
voltage-dependent 100-pS anion channel (mCS; mouse mitoplast, n = 5 patches) or of VDAC (yeast VDAC in liposomes, n = 3 patches; n = 1 mouse
mitochondria) (data not shown). In addition, no significant change in
current was observed upon introduction of targeting peptide to the bath
with azolectin patches without channel activity (n = 3
patches), although peptide addition sometimes resulted in loss of
patch-seal.
Targeting Peptide Effects Vary with Dose and
Voltage
The concentration range over which the COX-IV targeting
peptide caused transient blockade of MCC was 10-100 µM as shown in Fig. 3. In previous studies from other
laboratories, similar levels of targeting peptides, e.g. from
yeast COX-IV, reversibly inhibited protein
import(22, 23, 24) . The COX-VI targeting
peptide blocked MCC at lower concentrations (0.5-5
µM) in this study (not shown), and, unfortunately, the
effects of this peptide on import have not yet been reported. Similar
low concentrations (0.2 µM) of appropriate signal peptides
affected Escherichia coli, protein-conducting
channels(30) .
Figure 3:
Dose
response of yeast MCC activity to targeting peptide. The percent block
(or percent decrease) in open probability of MCC caused by varied
levels of COX-IV peptide is shown at -40 () and 40 mV
(
). Open probability is calculated from amplitude histograms (bin
width of 0.4 pA) as the fraction of total time spent in the fully open
state. MCC was recorded from an excised proteoliposome derived from
inner membranes isolated from yeast mitochondria strain M22-2
that lack VDAC. Other conditions are as described in the legends to
Figs. 1 and 2.
The effects of targeting peptide on the open
probability of MCC were prominent at positive but not negative
voltages. This is shown in Fig. 4B for COX-IV peptide
and mouse MCC and in Fig. 5for COX-IV peptide and yeast
MCC. The addition of targeting peptides resulted in an increase in
flicker rate (measured as mean open time) between the various
conductance levels at positive potentials. For example, the mean open
time of the 1-nS level of wild-type yeast MCC at 30 mV decreased
from 13.6 ± 3.4 (n = 3 patches) to 1.6 ±
0.2 ms (n = 3 patches) upon addition of the COX-IV
peptide. In contrast, there was no significant difference in mean open
times at -30 mV in the absence (15.6 ± 5.9 (n = 3)) and presence (11.3 ± 1.7 ms (n = 3)) of COX-IV peptide. Similarly, the COX-VI peptide
significantly altered the open probability and mean open time at
positive potentials. However, small decreases in both parameters were
noted at negative potentials with COX-VI peptide (data not shown).
Figure 4:
Voltage dependence of the effect of a
targeting peptide on mammalian MCC activity. A, current traces
at 30 mV from a patch excised from a mouse kidney mitoplast are shown
in the presence (bottom) and absence (top) of 50
µM COX-IV peptide. O and C correspond to
the open and closed conductance levels. B, the voltage
dependence of the open probability is shown in the absence () and
presence (
) of COX-IV peptide. The pipette and bath patching
medium also included 5 mM succinate, 0.2 mM
KH
PO
, 2 mM ADP, and 5 mM MgCl
. Other conditions are as described in the legends
to Figs. 1 and 2.
Figure 5:
Voltage dependence of the effect of a
targeting peptide on yeast MCC activity. The fractional change in open
probability (normalized to the control) is shown for the 1-nS
level with and without COX-IV peptide as a function of voltage for
wild-type (
) and VDAC-less (
) yeast MCC activity. Other
conditions as described in the legends to Figs.
1-3.
Relationship between MCC and Mitochondrial Protein
Import
The effects on MCC by the targeting peptides were
characterized by a dramatic yet reversible ``block'' of the
channel. These changes were similar to the increase in flickering and
decrease in open probability reported for another mitochondrial
channel, the peptide-sensitive channel (PSC). This cation-selective
channel, which has been localized to the outer
membrane(22, 26, 27, 28) , was
implicated in protein import by its sensitivity to similar levels of a
yeast COX-IV peptide (26, 27) initially in tip-dip
reconstitution studies and more recently in yeast
mitoplasts(29) . In the present study, a peptide-sensitive
activity was recorded from yeast outer membrane proteoliposomes. The
peptide-induced changes in PSC behavior were partially attributed to an
intermittent occlusion of the pore during the translocation of the
positively charged peptide into the pipette at bath negative
potentials(26, 27, 28) . Occlusion of the pore
is also thought to occur during protein translocation in the
endoplasmic reticulum(25) , E. coli plasma
membranes(30) , and chloroplasts(31) . The peptide
sensitivity of MCC, like that of PSC, is strongly suggestive of an
involvement of these channels in protein import. Further work must be
done to correlate these two activities and define their relationships
to known yeast import-apparatus proteins, e.g. inner and outer
membrane import ``pore'' proteins such as MIM23 and
MOM38(2, 3) .
-helixes in a suitable
environment(7) . As shown in , there may also be a
correlation between peptide structure and MCC blockade. Of the four
peptides tested, only the two targeting peptides are both cationic and
-helical (Ref. 21 and our results for COX-VI, not shown).
Conclusions
The open probability and mean open
time of yeast and mammalian MCC are decreased specifically by targeting
peptides from cytochrome oxidase subunit IV and VI. Two other
mitochondrial channel activities are unperturbed by one targeting
peptide, and MCC is not affected by two non-targeting peptides derived
from an outer membrane protein. Although other interpretations are
possible, these results support a role for MCC activity in the import
of mitochondrial inner membrane proteins.
Table: Synthetic peptide characteristics and effects on
MCC
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.