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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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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-IV1-13 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-IV1-13 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.
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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-IV1-13 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-IV1-13 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.

Mentions: 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).


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)

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-IV1-13 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-IV1-13 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139772&req=5

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-IV1-13 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-IV1-13 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.
Mentions: 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).

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