©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeting Peptides Transiently Block a Mitochondrial Channel (*)

Timothy A. Lohret (1) (3), Kathleen W. Kinnally (1) (2)(§)

From the (1)Division of Molecular Medicine, Wadsworth Center, Albany, New York 12201-0509 and the Departments of (2)Biomedical Sciences and (3)Biological Sciences, State University of New York, Albany, New York 12222

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 -helices and are involved in both recognition and translocation of these proteins(2, 7) .

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()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.

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.


EXPERIMENTAL PROCEDURES

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 (10M 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).


RESULTS AND DISCUSSION

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 KHPO, 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) .

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 -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



FOOTNOTES

*
This study was supported by National Science Foundation Grant MCB9117658. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Wadsworth Center, Empire State Plaza, P. O. Box 509, Albany, NY 12201-0509. Tel.: 518-474-4229; Fax: 518-474-7992; E-mail: Kinnally@wadsworth.org.

The abbreviations used are: nS, nanosiemens; pS, picosiemens; MCC, multiple conductance channel; PSC, peptide-sensitive channel; VDAC, voltage-dependent anion-selective channel; COX-IV, targeting peptide of subunit IV from cytochrome oxidase; COX-VI, targeting peptide of subunit VI from cytochrome oxidase; mCS, mitochondrial centum picosiemen.

T. A. Lohret and K. W. Kinnally, unpublished results.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.