Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC

Abstract

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.

Figure 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.

Figure 2

Presequence peptides rapidly induce a transient blockade of MCC conductance. The patch containing two MCC was excised with a micropipette from a proteoliposome prepared with mitochondrial inner membranes from the wild-type strain and the patch conductance was measured at 20 mV as described in Materials and Methods. The current trace shows the time course of the effect on the two channels of perfusing 3 ml of media containing 50 μM yCOX-IV_1-13 into the 0.5-ml bath solution. (A) The current trace shown was obtained using an Omniscribe recorder whose effective filtration rate is 0.05 kHz. This current trace was also analyzed using the PAT computer program at 2 kHz as detailed in the Materials and Methods section and 0.5-s regions show the current trace before (B), during (C), and after (D) the introduction of yCOX-IV_1-13 to the bath by perfusion on a faster time scale. Media were 0.15 M KCl, 5 mM Hepes, 1 mM EGTA, 1.05 mM CaCl₂, pH 7.4.

Figure 3

Presequence peptides specifically induce a transient blockade of MCC conductance. The conductance of a patch excised from a proteoliposome-containing mitochondrial inner membranes from the wild-type strain was measured at 20 mV. The single channel current traces in the absence of peptide (control) and in the presence of 50 μM synB2 or yCOX-IV_1-13 peptide in the bath solution were band-width limited to 2 kHz. Total current amplitude diagrams and current traces show the occupancy of open (O), substate (S) and closed (C) conductance levels. The probability of occupying the open state was 0.9, 0.8, and 0.4 in the absence of peptide (control), in the presence of synB2, and in the presence of yCOX-IV_1-13, respectively.

Figure 4

Tim23 antibodies specifically inhibit MCC activity. (A) Typical current traces at 40 mV recorded from proteoliposomes prepared from wild-type inner membranes that were preincubated with Tim23 IgG or preimmune IgG as detailed in Materials and Methods. (B) The fraction of patches in which MCC was detected (at voltages between ±60mV) in a blind study after incubation of proteoliposomes containing∼1 μg inner membrane protein and 25 μg of the indicated antibody. All studies were normalized to untreated proteoliposomes. n is the number of patches examined for each condition. (C) The fraction of patches in which the outer membrane channel activity PSC was detected (at voltages between ±60 mV) after incubation of proteoliposomes containing∼1 μg outer membrane protein and 25 μg of the indicated antibody was normalized to untreated proteoliposomes.

Figure 4

Tim23 antibodies specifically inhibit MCC activity. (A) Typical current traces at 40 mV recorded from proteoliposomes prepared from wild-type inner membranes that were preincubated with Tim23 IgG or preimmune IgG as detailed in Materials and Methods. (B) The fraction of patches in which MCC was detected (at voltages between ±60mV) in a blind study after incubation of proteoliposomes containing∼1 μg inner membrane protein and 25 μg of the indicated antibody. All studies were normalized to untreated proteoliposomes. n is the number of patches examined for each condition. (C) The fraction of patches in which the outer membrane channel activity PSC was detected (at voltages between ±60 mV) after incubation of proteoliposomes containing∼1 μg outer membrane protein and 25 μg of the indicated antibody was normalized to untreated proteoliposomes.

Figure 4

Tim23 antibodies specifically inhibit MCC activity. (A) Typical current traces at 40 mV recorded from proteoliposomes prepared from wild-type inner membranes that were preincubated with Tim23 IgG or preimmune IgG as detailed in Materials and Methods. (B) The fraction of patches in which MCC was detected (at voltages between ±60mV) in a blind study after incubation of proteoliposomes containing∼1 μg inner membrane protein and 25 μg of the indicated antibody. All studies were normalized to untreated proteoliposomes. n is the number of patches examined for each condition. (C) The fraction of patches in which the outer membrane channel activity PSC was detected (at voltages between ±60 mV) after incubation of proteoliposomes containing∼1 μg outer membrane protein and 25 μg of the indicated antibody was normalized to untreated proteoliposomes.

Figure 5

The conductance of MCC isolated from the tim23-1 mutant is not blocked by presequence peptides in the bath. Proteoliposomes were prepared from inner membranes from wildtype cells (A) or the tim23-1 mutant (B), and current traces of MCC activity were recorded from patches in the presence and absence of 50 μM yCOX-IV_1-13 peptide in the bath solution. (C) The flicker rate (events/sec) from the open state to lower conductance states of MCC at 20 mV from wild-type and tim23-1 strains was determined in the absence (control) and presence of 50 μM of either yCOX-IV_1-13 or fCOX-IV_3-22. Error bars indicate standard deviations from a minimum of four determinations.

Figure 5

The conductance of MCC isolated from the tim23-1 mutant is not blocked by presequence peptides in the bath. Proteoliposomes were prepared from inner membranes from wildtype cells (A) or the tim23-1 mutant (B), and current traces of MCC activity were recorded from patches in the presence and absence of 50 μM yCOX-IV_1-13 peptide in the bath solution. (C) The flicker rate (events/sec) from the open state to lower conductance states of MCC at 20 mV from wild-type and tim23-1 strains was determined in the absence (control) and presence of 50 μM of either yCOX-IV_1-13 or fCOX-IV_3-22. Error bars indicate standard deviations from a minimum of four determinations.

Figure 5

The conductance of MCC isolated from the tim23-1 mutant is not blocked by presequence peptides in the bath. Proteoliposomes were prepared from inner membranes from wildtype cells (A) or the tim23-1 mutant (B), and current traces of MCC activity were recorded from patches in the presence and absence of 50 μM yCOX-IV_1-13 peptide in the bath solution. (C) The flicker rate (events/sec) from the open state to lower conductance states of MCC at 20 mV from wild-type and tim23-1 strains was determined in the absence (control) and presence of 50 μM of either yCOX-IV_1-13 or fCOX-IV_3-22. Error bars indicate standard deviations from a minimum of four determinations.

Figure 6

Rapid flickering induced by presequence peptides in the micropipette solution is reduced in MCC from the tim23-1 mutant. Patches were excised from proteoliposomes containing wild-type (A) or tim23-1 (B) mitochondrial inner membranes. Typical current traces of MCC activity at −20 mV recorded from different patches in the presence and absence of 100 μM yCOXIV_1-13 peptide in the micropipette are shown. P_O corresponds to probability of occupying the open state.

Figure 6

Rapid flickering induced by presequence peptides in the micropipette solution is reduced in MCC from the tim23-1 mutant. Patches were excised from proteoliposomes containing wild-type (A) or tim23-1 (B) mitochondrial inner membranes. Typical current traces of MCC activity at −20 mV recorded from different patches in the presence and absence of 100 μM yCOXIV_1-13 peptide in the micropipette are shown. P_O corresponds to probability of occupying the open state.

Figure 7

Dose dependence of blockade of MCC conductance by yCOX-IV_1-13 peptide. Flicker rates for typical MCC activity from wild-type (•)or tim23-1 (○) strains at different concentrations of the yCOX-IV_1-13 peptide in the bath were determined from current traces (5–15 s) at 20 mV in single channel patches.

Figure 8

Mitochondria isolated from the tim23-1 mutant contain reduced amounts of the Tim23-1 protein. Mitochondria were isolated from wild-type cells and the tim23-1 mutant, and proteoliposomes were prepared as described in Materials and Methods. Aliquots representing 30 μg of proteoliposomes were immune blotted and decorated with antibodies to the Tim23 protein (Tim23p) and to cytochrome oxidase subunit IV (COX4p).

Figure 9

The tim23-1 mutation does not affect the peptide-sensitive channel, PSC, of the mitochondrial outer membrane. (A) Sample current traces for PSC from proteoliposomes containing outer membranes from wild-type or tim23-1 strains are shown in the presence and absence (control) of 50 μM yCOX-IV_1-13 peptide in the bath. (B) The flicker rates of PSC of wild-type and tim23-1 strains were determined from current traces represented by those in A above.

Figure 9

The tim23-1 mutation does not affect the peptide-sensitive channel, PSC, of the mitochondrial outer membrane. (A) Sample current traces for PSC from proteoliposomes containing outer membranes from wild-type or tim23-1 strains are shown in the presence and absence (control) of 50 μM yCOX-IV_1-13 peptide in the bath. (B) The flicker rates of PSC of wild-type and tim23-1 strains were determined from current traces represented by those in A above.