Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139772&req=5

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.

Mentions: Mitochondria were isolated from wild-type strain AH216 and the tim23-1 mutant as described (Emtage and Jensen, 1993; Daum et al., 1982). Mitochondrial membranes were prepared by the French press method (Decker and Greenawalt, 1977), and the outer membrane was separated from the inner membrane as described by Mannella (1982). The purity of the membrane fractions was assayed in two ways. First, immunoblotting showed, for the most part, that the outer membrane protein, voltage-dependent anion-selective channel (VDAC), was found only in the outer membrane preparations, and that the inner membrane protein Tim23 was found solely in the inner membrane preparation (see Fig. 1). Second, we found that VDAC activity detected by patch-clamp analysis was found only in outer membrane, but not in inner membrane preparations (Lohret and Kinnally, 1995a; Lohret et al., 1996). Inner and outer membranes were separately reconstituted into giant proteoliposomes (Sigma Type IV-S soybean l-α-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).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Mitochondria were isolated from wild-type strain AH216 and the tim23-1 mutant as described (Emtage and Jensen, 1993; Daum et al., 1982). Mitochondrial membranes were prepared by the French press method (Decker and Greenawalt, 1977), and the outer membrane was separated from the inner membrane as described by Mannella (1982). The purity of the membrane fractions was assayed in two ways. First, immunoblotting showed, for the most part, that the outer membrane protein, voltage-dependent anion-selective channel (VDAC), was found only in the outer membrane preparations, and that the inner membrane protein Tim23 was found solely in the inner membrane preparation (see Fig. 1). Second, we found that VDAC activity detected by patch-clamp analysis was found only in outer membrane, but not in inner membrane preparations (Lohret and Kinnally, 1995a; Lohret et al., 1996). Inner and outer membranes were separately reconstituted into giant proteoliposomes (Sigma Type IV-S soybean l-α-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).

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