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

Lohret TA, Jensen RE, Kinnally KW - J. Cell Biol. (1997)

Bottom Line: We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University at Albany, SUNY, New York 12222, USA.

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.

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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.
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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.

Mentions: 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).


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

Lohret TA, Jensen RE, Kinnally KW - J. Cell Biol. (1997)

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.
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Related In: Results  -  Collection

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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.
Mentions: 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).

Bottom Line: We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University at Albany, SUNY, New York 12222, USA.

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.

Show MeSH
Related in: MedlinePlus