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© The Rockefeller University Press,
0021-9525/1997//377 $5.00
The Journal of Cell Biology, Volume 137, Number 2,
, 1997 377-386
Article |
Tim23, a Protein Import Component of the Mitochondrial Inner Membrane, Is Required for Normal Activity of the Multiple Conductance Channel, MCC
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). 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.
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; 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).
Tim23 is an integral protein of the inner membrane essential for import (Emtage and Jensen, 1993; 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.
Both mitochondrial membranes contain a number of channel activities which have been identified using electrophysiological techniques (Kinnally et al., 1992; 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).
We have recently shown that the conductance of MCC is transiently blocked by synthetic peptides corresponding to mitochondrial presequences (Lohret and Kinnally, 1995b). 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.
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Materials and Methods |
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-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).
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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).
Antibody Inhibition
Preimmune IgG and IgG against Tim23p were prepared from serum as described (Emtage and Jensen, 1993). 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.
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). 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.
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Results |
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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).
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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 β-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).
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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.
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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).
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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.
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Discussion |
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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.
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Acknowledgments |
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Submitted: 6 June 1996
Revised: 22 January 1997
1. Abbreviations used in this paper: MCC, multiple conductance channel; PSC, peptide-sensitive channel; VDAC, voltage-dependent anion-selective channel.
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) strains at different concentrations of the yCOX-IV1-13 peptide in the bath were determined from current traces (5–15 s) at 20 mV in single channel patches.
