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Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria.

Davis AJ, Sepuri NB, Holder J, Johnson AE, Jensen RE - J. Cell Biol. (2000)

Bottom Line: Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import.In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear.The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p-Tim13p and Tim9p-Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

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The Tim8p–Tim13p complex binds to the NH2 terminus of Tim23p. (A) Tim23Δ2-94, a Tim23p construct lacking its first 94 residues, no longer cross-links to Tim8p and Tim13p. Top panel shows a schematic representation of the Tim23 protein and the Tim23Δ2-94 construct. The numbered rectangles represent the membrane-spanning segments within Tim23p. Small numbers above the figures correspond to amino acid numbers within Tim23p. In the bottom panel, 35S-labeled Tim23Δ2-94 was imported into mitochondria lacking IM potential and treated with SMPB or no cross-linker (−XL). An aliquot of mitochondria (50 μg) was pelleted (SMPB or −XL). Aliquots containing 200 μg of mitochondria were solubilized, immunoprecipitated with antiserum to either Tim8p, Tim9p, Tim10p, or Tim13p, and then analyzed by SDS-PAGE and phosphorimaging. Black arrowhead identifies cross-links. (B) Fusion of the Tim23p NH2-terminal domain to Aac2p recruits Tim8p and Tim13p binding. The top panel shows schematic representations of the Aac2 protein and Tim23N-Aac2 fusion protein. The numbered rectangles correspond to membrane-spanning segments within Aac2p. Small numbers above the black figure correspond to the amino acid number of Aac2p. The white rectangle represents residues 1–96 of the Tim23p NH2 terminus. In the bottom panel, 35S-labeled Aac2p and Tim23N-Aac2p were imported into mitochondria in the absence of a membrane potential, cross-linked with SMPB, immune precipitated, and analyzed as described above. Black arrowheads identify cross-links.
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Figure 4: The Tim8p–Tim13p complex binds to the NH2 terminus of Tim23p. (A) Tim23Δ2-94, a Tim23p construct lacking its first 94 residues, no longer cross-links to Tim8p and Tim13p. Top panel shows a schematic representation of the Tim23 protein and the Tim23Δ2-94 construct. The numbered rectangles represent the membrane-spanning segments within Tim23p. Small numbers above the figures correspond to amino acid numbers within Tim23p. In the bottom panel, 35S-labeled Tim23Δ2-94 was imported into mitochondria lacking IM potential and treated with SMPB or no cross-linker (−XL). An aliquot of mitochondria (50 μg) was pelleted (SMPB or −XL). Aliquots containing 200 μg of mitochondria were solubilized, immunoprecipitated with antiserum to either Tim8p, Tim9p, Tim10p, or Tim13p, and then analyzed by SDS-PAGE and phosphorimaging. Black arrowhead identifies cross-links. (B) Fusion of the Tim23p NH2-terminal domain to Aac2p recruits Tim8p and Tim13p binding. The top panel shows schematic representations of the Aac2 protein and Tim23N-Aac2 fusion protein. The numbered rectangles correspond to membrane-spanning segments within Aac2p. Small numbers above the black figure correspond to the amino acid number of Aac2p. The white rectangle represents residues 1–96 of the Tim23p NH2 terminus. In the bottom panel, 35S-labeled Aac2p and Tim23N-Aac2p were imported into mitochondria in the absence of a membrane potential, cross-linked with SMPB, immune precipitated, and analyzed as described above. Black arrowheads identify cross-links.

Mentions: To determine where the Tim8 and Tim13 proteins bind within Tim23p, we examined the import of different Tim23p constructs. As shown previously, all of the information required to import Tim23p into isolated mitochondria is carried in the COOH-terminal half of the protein (Davis et al. 1998). Therefore, we imported Tim23Δ2-94, which lacks the first 94 residues of Tim23p, into mitochondria in the absence of an IM potential, and added the cross-linker SMPB. Immune precipitations showed that Tim23Δ2-94 cross-linked to both Tim9p and Tim10p, but no detectable cross-links to Tim8p and Tim13p were formed (Fig. 4 A). Our results indicate that the NH2-terminal region of the Tim23 protein is required for interactions with Tim8p and Tim13p, whereas Tim9p and Tim10p interact with the COOH terminus of Tim23p.


Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria.

Davis AJ, Sepuri NB, Holder J, Johnson AE, Jensen RE - J. Cell Biol. (2000)

The Tim8p–Tim13p complex binds to the NH2 terminus of Tim23p. (A) Tim23Δ2-94, a Tim23p construct lacking its first 94 residues, no longer cross-links to Tim8p and Tim13p. Top panel shows a schematic representation of the Tim23 protein and the Tim23Δ2-94 construct. The numbered rectangles represent the membrane-spanning segments within Tim23p. Small numbers above the figures correspond to amino acid numbers within Tim23p. In the bottom panel, 35S-labeled Tim23Δ2-94 was imported into mitochondria lacking IM potential and treated with SMPB or no cross-linker (−XL). An aliquot of mitochondria (50 μg) was pelleted (SMPB or −XL). Aliquots containing 200 μg of mitochondria were solubilized, immunoprecipitated with antiserum to either Tim8p, Tim9p, Tim10p, or Tim13p, and then analyzed by SDS-PAGE and phosphorimaging. Black arrowhead identifies cross-links. (B) Fusion of the Tim23p NH2-terminal domain to Aac2p recruits Tim8p and Tim13p binding. The top panel shows schematic representations of the Aac2 protein and Tim23N-Aac2 fusion protein. The numbered rectangles correspond to membrane-spanning segments within Aac2p. Small numbers above the black figure correspond to the amino acid number of Aac2p. The white rectangle represents residues 1–96 of the Tim23p NH2 terminus. In the bottom panel, 35S-labeled Aac2p and Tim23N-Aac2p were imported into mitochondria in the absence of a membrane potential, cross-linked with SMPB, immune precipitated, and analyzed as described above. Black arrowheads identify cross-links.
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Figure 4: The Tim8p–Tim13p complex binds to the NH2 terminus of Tim23p. (A) Tim23Δ2-94, a Tim23p construct lacking its first 94 residues, no longer cross-links to Tim8p and Tim13p. Top panel shows a schematic representation of the Tim23 protein and the Tim23Δ2-94 construct. The numbered rectangles represent the membrane-spanning segments within Tim23p. Small numbers above the figures correspond to amino acid numbers within Tim23p. In the bottom panel, 35S-labeled Tim23Δ2-94 was imported into mitochondria lacking IM potential and treated with SMPB or no cross-linker (−XL). An aliquot of mitochondria (50 μg) was pelleted (SMPB or −XL). Aliquots containing 200 μg of mitochondria were solubilized, immunoprecipitated with antiserum to either Tim8p, Tim9p, Tim10p, or Tim13p, and then analyzed by SDS-PAGE and phosphorimaging. Black arrowhead identifies cross-links. (B) Fusion of the Tim23p NH2-terminal domain to Aac2p recruits Tim8p and Tim13p binding. The top panel shows schematic representations of the Aac2 protein and Tim23N-Aac2 fusion protein. The numbered rectangles correspond to membrane-spanning segments within Aac2p. Small numbers above the black figure correspond to the amino acid number of Aac2p. The white rectangle represents residues 1–96 of the Tim23p NH2 terminus. In the bottom panel, 35S-labeled Aac2p and Tim23N-Aac2p were imported into mitochondria in the absence of a membrane potential, cross-linked with SMPB, immune precipitated, and analyzed as described above. Black arrowheads identify cross-links.
Mentions: To determine where the Tim8 and Tim13 proteins bind within Tim23p, we examined the import of different Tim23p constructs. As shown previously, all of the information required to import Tim23p into isolated mitochondria is carried in the COOH-terminal half of the protein (Davis et al. 1998). Therefore, we imported Tim23Δ2-94, which lacks the first 94 residues of Tim23p, into mitochondria in the absence of an IM potential, and added the cross-linker SMPB. Immune precipitations showed that Tim23Δ2-94 cross-linked to both Tim9p and Tim10p, but no detectable cross-links to Tim8p and Tim13p were formed (Fig. 4 A). Our results indicate that the NH2-terminal region of the Tim23 protein is required for interactions with Tim8p and Tim13p, whereas Tim9p and Tim10p interact with the COOH terminus of Tim23p.

Bottom Line: Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import.In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear.The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p-Tim13p and Tim9p-Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

Show MeSH
Related in: MedlinePlus