1. Tim23 Links the Inner and Outer Mitochondrial Membranes
Mariel Donzeau, Krisztina Káldi, Alexander Adam, Stefan Paschen, Gerhard Wanner, Bernard Guiard, Matthias F Bauer, Walter Neupert, Michael Brunner 
Cell 
Volume 101, Issue 4, Pages (May 2000)
DOI: /S (00)

2. Figure 1
The N-Terminal Domain of Tim23 Is Required for Growth of Yeast at 37°C
(A) Tim23 and Tim23Δ50 are schematically outlined. Tim23 consists of 222 amino acid residues. Its C-terminal half (gray box) is integrated into the inner membrane. Tim23Δ50 is an N-terminally truncated protein that starts at amino acid residue 51. Haploid YPH501 (tim23::URA3) cells harboring the low copy number plasmid pFL59-Tim23 and pFL59-Tim23Δ50, respectively, were grown at 25°C in selective medium containing 2% glycerol to OD600 = 1.5. The cell cultures were subjected to consecutive 10-fold dilution steps, and 3 μl aliquots of each dilution were spotted on glycerol-containing agar plates that were incubated at 25°C and 37°C.
(B) MB2-Tim23 cells and MB2-Tim23Δ50his12 cells (Bauer et al. 1996) were grown at 25°C in selective medium containing 2% galactose to OD600 = 0.08 and then incubated at 37°C. Aliquots were removed after the indicated time periods, and OD600 was determined. Cell number at 0 hr at 37°C was set equal to 1.
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3. Figure 2
The N-Terminal Domain of Tim23 Facilitates Efficient Precursor Import into Mitochondria
(A) Import of the ADP/ATP carrier. Mitochondria were prepared from yeast strains MB2-Tim23 and MB2-Tim23Δ50his12. Energized mitochondria (30 μg) were incubated for 3 min at 25°C with radiolabeled AAC precursor. When indicated, samples were treated with 100 μg/ml PK or mitoplasts were generated by swelling (Sw) and treated with PK. Samples were analyzed by SDS-PAGE and autoradiography. AAC′, proteolytic fragment of inserted AAC.
(B) Import of chemical amounts of pSu9(1-69)DHFR. Mitochondria and mitoplasts (30 μg) were incubated at 25°C for the indicated time periods with 30 pmol purified recombinant pSu9(1-69)DHFR and trace amounts of 35S-labeled pSu9(1-69)DHFR. Samples were treated with PK and subjected to SDS-PAGE, and the mature form of Su9(1-69)DHFR was quantified with a phosphorimaging system. 100 arb. units correspond to approximately 1.5 pmol protein.
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4. Figure 3
The N Terminus of Tim23 Is Accessible to PK in Intact Mitochondria
(A) Mitochondria (M) and mitoplasts (MP) were incubated for 20 min on ice with 300 and 50 μg/ml PK, respectively. Samples were analyzed by SDS-PAGE and immunoblotting with affinity-purified antibodies against the C terminus of Tim23, α23C (upper panel), and antibodies against the first half of Tim23, α23 (lower panel). Tim23*, proteolytic fragment of Tim23 in mitochondria. Tim23C-term, C-terminal portion of Tim23 integrated into the inner membrane.
(B) Mitochondria and mitoplasts were treated with PK and analyzed using antibodies against cytochrome b2 (Cyt b2), AAC, and Tim17.
(C) Mapping of the PK cleavage site in Tim23 in intact mitochondria. Tim23Δ10 and Tim23Δ20, which lack 10 and 20 N-terminal amino acid residues, respectively, and full-size Tim23 were synthesized in vitro in the presence of 35S methionine. The precursors were then incubated for 15 min at 25°C with energized mitochondria. Samples were diluted 10-fold in HEPES-sorbitol buffer, treated with 200 μg/ml PK when indicated, and analyzed by SDS-PAGE and autoradiography. The fragment generated by PK is indicated by an asterisk.
(D) Mitochondria were incubated with NADH (+Δψ) and CCCP (-Δψ) and then treated with 200 μg/ml PK.
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5. Figure 4 All Tim23 Is Accessible to PK in Intact Mitochondria
(A) Mitochondria were incubated for the indicated time periods at 0°C with 1 mg/ml PK.
(B) Mitochondria were treated for 20 min with 0.5 mg/ml PK at the indicated temperatures.
(C) Mitoplasts (MP) and mitochondria (M) were treated for 30 min at 0°C with the indicated concentrations of PK. Tim23* is indicated by an asterisk. Samples were analyzed by SDS-PAGE and immunoblotting with affinity-purified antibodies against Tim23.
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6. Figure 5
The Accessibility of Tim23 in Mitochondria Does Not Correlate with the Number of TOM Complexes
(A) Cells harboring wild-type TOM40 (WT) and cells harboring a temperature-sensitive allele of TOM40 (ts1) (Kassenbrock et al. 1993) were grown at 25°C and then shifted to 37°C for 6 hr. Mitochondria were prepared and treated for 20 min at 0°C with 0.2 mg/ml PK when indicated.
(B) The Gal10-TOM40 integration cassette is schematically outlined (the Experimental Procedures). W334 cells were transformed with the cassette to replace by homologous recombination the TOM40 promoter in the chromosome by the Gal10 promoter. Transformants were grown in selective lactate medium containing 1% galactose (+gal) and shifted, when indicated, for 12 hr to galactose-free growth medium (-gal) before mitochondria were prepared. Mitochondria were treated with 0.2 mg/ml PK (lower panels). Samples were analyzed with antibodies against Tom40 and Tim23.
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7. Figure 6
The N-Terminal Domain of Tim23 Is Inserted into the Outer Membrane
(A) Import of Ig-Tim23(1-73) into the outer membrane in the absence of Δψ. Ig-Tim23(1-73), a fusion protein of the CH2-CH3 domains of human IgG (Ig) and the N-terminal 73 amino acid residues of Tim23, was incubated for 15 min at 25°C with mitochondria in the presence of valinomycin to dissipate Δψ. The mitochondria were then treated with 50 μg/ml trypsin and 1 M NaCl and reisolated by centrifugation. Supernatant (S) and pellet (P) fractions were analyzed.
(B) Ig-Tim23(1-73) was imported into mitochondria. Samples were treated with trypsin and 1 M NaCl or washed with 6 M urea, and mitochondria were reisolated. Samples were analyzed by SDS-PAGE, and Ig-Tim23(1-73) was quantified with a phosphorimaging system. 100% corresponds to the total amount of precursor associated with the mitochondria.
(C) Mitochondria, pretreated with or without 100 μg/ml trypsin to remove the surface receptors, were reisolated and incubated for 15 min at 25°C with radiolabeled Ig-Tim23(1-73). The samples were halved; one half was left untreated (“total”) and the other half was incubated with 1 M NaCl and treated with trypsin (“imported”). Ig-Tim23(1-73) associated with mitochondria was quantified.
(D) Tim23(1-62)-DHFR is imported into the outer membrane in vivo. Mitochondria were isolated from yeast cells expressing Tim23(1-62)-DHFR (Káldi et al. 1998). Submitochondrial particles were generated and isolated by two consecutive sucrose density gradient centrifugations (the Experimental Procedures). Aliquots were analyzed by SDS-PAGE and immunoblotting for the distribution of the indicated antigens. Subunit g, integral membrane subunit of the F0 segment of the ATP synthase; IDF + IM, intermediate density fraction and inner membrane vesicles; OMV, outer membrane vesicles.
(E) Tim23(1-62)DHFR is an integral membrane protein. Mitochondria harboring Tim23(1-62)DHFR were extracted with carbonate (pH 11). Mitochondrial membranes were separated from the extracted proteins by flotation centrifugation in a sucrose gradient, and the fractions were analyzed by SDS-PAGE and immunoblotting. F1β, β-subunit of the F1 segment of the ATP synthase.
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8. Figure 7 Submitochondrial Topology of Tim23 Fusion Proteins
(A) The CH2-CH3 domains of Ig-Tim23 but not of Tim23Δ50 are accessible to PK in intact mitochondria. Ig-Tim23 and Ig-Tim23Δ50, fusion proteins of the CH2-CH3 domains with Tim23 and Tim23Δ50, respectively, are schematically outlined in the upper parts. Left panel, total protein extract from Ig-Tim23 cells (total). Mitochondria were prepared and treated with the indicated concentrations of PK, or mitoplasts were generated (SW) and treated with PK. Samples were analyzed by SDS-PAGE and immunoblotting using affinity-purified antibodies specific for the C terminus of Tim23. **, proteolytic fragment of Ig-Tim23 generated during preparation of mitochondria. Tim23*, fragment of Ig-Tim23 generated by PK treatment of intact mitochondria. Tim23C-term, 13 kDa PK-resistant C-terminal portion of Tim23 that is integrated into the inner membrane. Right panel, mitochondria from Ig-Tim23Δ50 and from WT cells were treated with PK when indicated and analyzed by immunoblotting with α23. Tim23* is indicated by an asterisk. The mobility of size standards is indicated; numbers correspond to molecular mass in kDa.
(B) The CH2-CH3 domains of Ig-Tim23 are exposed on the surface of the mitochondria. Mitochondria from WT cells and Ig-Tim23 cells were incubated with protein A conjugated with 10 nm colloidal gold particles, and samples were processed for electron microscopy.
(C) Quantification of surface labeling of mitochondria with protein A-gold. The number of gold particles associated with 50 mitochondrial particles from WT cells, Ig-Tim23 cells, and Ig-Tim23Δ50 cells were determined.
(D) Ig-Tim23 and Ig-Tim23Δ50 are integral membrane proteins. Mitochondria from WT, Ig-Tim23, and Ig-Tim23Δ50 cells were extracted with 100 mM Na2CO3 (pH 11), and membranes were reisolated by centrifugation. Tim23 and Tim23 fusion proteins in the pellet and supernatant fractions were analyzed by SDS-PAGE and Western blotting and quantified by densitometry.
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9. Figure 8
Model for the Transfer of a Precursor from the TOM Complex to the TIM23 Complex
(1) The TIM23 complex is a dimer, containing two molecules of Tim23 (23), Tim17 (17), and Tim44 (44), which provides two binding sites for mt-Hsp70 (70). Only one molecule of mt-Hsp70 is shown. Tim23 spans both mitochondrial membranes. The N-terminal domain of Tim23 is integrated into the outer membrane (OM). Residues 50 to 100 dimerize and form a negatively charged presequence receptor domain in the intermembrane space (Bauer et al. 1996). The C-terminal half of Tim23 is integrated into the inner membrane (IM). A precursor in association with the TOM complex is shown. The positively charged matrix-targeting signal (zigzag) is bound to the trans-site (hatched). The TIM23 complex, tethered to the outer membrane via its N-terminal domain, screens by lateral diffusion the inner side of the outer membrane.
(2) The presequence receptor domain of Tim23 encounters the presequence and triggers its release from the trans-site of the TOM complex.
(3) Binding of the presequence leads to the Δψ-dependent opening of the protein-conducting channel of the TIM23 complex, and the presequence is translocated across the inner membrane.
(4) Further translocation is driven by ATP-dependent reaction cycles of mt-Hsp70, Tim44, and Mge1p (E) (Moro et al. 1999; Bauer et al. 2000).
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