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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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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).
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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).

Mentions: tim23-1 mutants are temperature-sensitive for growth and mitochondrial protein import, but mitochondria isolated from tim23-1 cells are defective in protein import in vitro at all temperatures (Emtage and Jensen, 1993). We have recently found that the Tim23-1 protein is rapidly degraded during mitochondrial isolation from tim23-1 strains (Ryan, K.R., R. Leung, and R.E. Jensen, manuscript submitted for publication). Similarly, we find that proteoliposomes prepared from tim23-1 inner membranes contain greatly reduced levels of the Tim23-1 protein (Fig. 8). Equal amounts of proteoliposomes prepared from wildtype and tim23-1 mitochondrial inner membranes were analyzed by immune blotting. While similar levels of cytochrome oxidase subunit IV (Cox4p; Fig. 8) and the β-subunit of the F1-ATPase (not shown) were seen in the two preparations, the level of the Tim23 protein was reduced at least 10-fold in the tim23-1 proteoliposomes. Importantly, the frequency of detecting MCC in proteoliposomes prepared from similar quantities of wild-type and tim23-1 inner membrane protein was virtually the same. Twelve MCC were detected in thirteen patches from proteoliposomes prepared with 16 μg tim23-1 inner membrane protein/mg lipid, while 0, 7, and 32 MCC were recorded from fifteen patches each from proteoliposomes prepared with 3, 27, and 133 μg wild-type inner membrane protein/ mg lipid, respectively. Hence, the reduced level of Tim23 protein in the inner membrane of tim23-1 mitochondria may explain their defect in protein import, as well as the alteration (but not loss) of MCC activity.


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)

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).
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Related In: Results  -  Collection

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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).
Mentions: tim23-1 mutants are temperature-sensitive for growth and mitochondrial protein import, but mitochondria isolated from tim23-1 cells are defective in protein import in vitro at all temperatures (Emtage and Jensen, 1993). We have recently found that the Tim23-1 protein is rapidly degraded during mitochondrial isolation from tim23-1 strains (Ryan, K.R., R. Leung, and R.E. Jensen, manuscript submitted for publication). Similarly, we find that proteoliposomes prepared from tim23-1 inner membranes contain greatly reduced levels of the Tim23-1 protein (Fig. 8). Equal amounts of proteoliposomes prepared from wildtype and tim23-1 mitochondrial inner membranes were analyzed by immune blotting. While similar levels of cytochrome oxidase subunit IV (Cox4p; Fig. 8) and the β-subunit of the F1-ATPase (not shown) were seen in the two preparations, the level of the Tim23 protein was reduced at least 10-fold in the tim23-1 proteoliposomes. Importantly, the frequency of detecting MCC in proteoliposomes prepared from similar quantities of wild-type and tim23-1 inner membrane protein was virtually the same. Twelve MCC were detected in thirteen patches from proteoliposomes prepared with 16 μg tim23-1 inner membrane protein/mg lipid, while 0, 7, and 32 MCC were recorded from fifteen patches each from proteoliposomes prepared with 3, 27, and 133 μg wild-type inner membrane protein/ mg lipid, respectively. Hence, the reduced level of Tim23 protein in the inner membrane of tim23-1 mitochondria may explain their defect in protein import, as well as the alteration (but not loss) of MCC activity.

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