Volume 87, Issue 1, Pages 33-41 (October 1996) Role of Tim23 as Voltage Sensor and Presequence Receptor in Protein Import into Mitochondria  Matthias F Bauer, Christian Sirrenberg, Walter Neupert, Michael Brunner  Cell  Volume 87, Issue 1, Pages 33-41 (October 1996) DOI: 10.1016/S0092-8674(00)81320-3

Figure 1 Functional Characterization of the N-Terminal Domain of Tim23 (A) Tim23 and Tim23-derived N-terminal deletion proteins. The structures of Tim23, Tim23his12, and the N-terminal deletion constructs Tim23Δ50his12 and Tim23Δ94his12 are schematically outlined. The hydrophilic N-terminal domain of Tim23 consisting of residues 1–101 (dark box) and the membrane integrated, hydrophobic portion corresponding to residues 102–222 (light box) are shown. The C-terminal 12his-tag is indicated by a black box. (B) The second half of the N-terminal domain of Tim23 is essential for the viability of yeast. Expression and mitochondrial localization of the Tim23 constructs in the diploid yeast strain MB2–23/Δ23 and their ability to complement a tim23::URA3 disruption are indicated. (C) Tim23his12 and Tim23Δ50his12 contribute to protein import. Upper panel: mitochondria, prepared from wild-type or yeast cells that express Tim23 together with Tim23his12 or Tim23Δ50his12 were analyzed by SDS–PAGE and Western blotting with antibodies against Tim23. Lower panel: energized mitochondria (50 μg) were incubated for 3 min at 25°C with purified radiolabeled pSu9(1–69)DHFR (100 ng). The samples were halved, one half was kept on ice (−PK) and the other half was treated with 100 μg/ml proteins K (+PK). Import was determined by SDS–PAGE and fluorography. p, i, m: precursor-, intermediate-, and mature-sized (respectively) forms of pSu9(1–69)DHFR. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 2 The N-Terminal Hydrophilic Domain of Tim23 Has the Potential to Dimerize (A) Fusion proteins between N-terminal segments of Tim23 and glutathion S-transferase (GST). Residues 1–101, 1–53, and 50–101 of Tim23 (dark boxes) were fused to GST (light box). The fusion proteins contain a 6his-tag at the N-terminus (black box). (B) Analysis by Superose 12 gel filtration of Tim23-GST fusion proteins. Constructs shown in (A) were expressed in E. coli and purified on Ni-NTA columns. Gel filtration of purified proteins (40 μg) is described in Experimental Procedures. Elution of size calibration standards is indicated by arrows. (C) Dimerization of Tim23-GST fusion proteins monitored by chemical cross-linking. The purified fusion proteins Tim23(1–101)GST, Tim23(1–53)GST, and Tim23(51–101)GST (5 μg/ml) were incubated in 30 mM HEPES–KOH (pH 7.4), 150 mM NaCl with 10 μM DFDNB for 30 min at 0°C. The cross-linker was quenched with glycine; TCA precipitation was performed followed by SDS-PAGE and immunoblotting with antibodies against Tim23. m, monomeric species; d, dimeric species. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 3 Tim23 Dimers Can Be Cross-Linked in the Mitochondrial Inner Membrane (A) Cross-linking of various forms of Tim23 constructs expressed in wild-type yeast. Mitoplasts (250 μg/ml) were subjected to cross-linking with 75 μM DSG. Samples were analyzed by SDS–PAGE and immunoblotting with antibodies against Tim23. Abbreviations: 23, Tim23 or Tim23his12; Δ50, Tim23Δ50his12. Bands corresponding in size to cross-links between Tim23 species are indicated. Asterisk: cross-link between Tim23 and an unidentified component of the TIM complex. (B) Identification of cross-linked Tim23 dimers by combined analysis with Ni-NTA affinity chromatography and immunodetection. Mitoplasts from yeast cells expressing Tim23 together with Tim23 Δ50his12 were subjected to cross-linking with DSG. Aliquots were directly analyzed by SDS–PAGE and immunoblotting with antibodies against full-length Tim23 (1–222) or with antibodies directed against the initial 53 amino acid residues of Tim23 (1–53). After cross-linking, the mitoplasts were dissolved in a Triton X-100 containing buffer and applied to a Ni-NTA column (Berthold et al. 1995). Bound proteins were eluted using an imidazole gradient. Main fractions were pooled, TCA precipitated, and subjected to SDS–PAGE followed by Western blotting. Immunodetection was performed with antibodies against full-length Tim23(1–222) or with antibodies against N-terminal portion of Tim23 (1–53). Tim23 and Tim23Δ50his12 are schematically outlined as shown in Figure 1A and cross-linked species are indicated. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 4 Cross-Linking of Tim23 Dimers Depends on the Membrane Potential Δω (A) Detection of Tim23 dimers in the presence of respiratory substrates and uncouplers. Mitochondria from Tim23his12 expressing wild-type cells were incubated with 75 μM DSG after the following additions: Control (no additions), NADH (4 mM), EtOH (90 mM ethanol), CCCP (0.1 mM carboxycyanide-m-chloro-phenylhydrazone), Val/Oli (0.1 μM valinomycin, 0.1 μM oligomycin), FCCP (25 μM carboxycyanide-p-trifluoromethoxy-phenylhydrazone), DNP (50 μM dinitrophenol), SF6487 (10 μM 3,5-(t-butyl)-4-hydroxybenzylidene-malononitrile). Samples were subjected to SDS–PAGE and Western blotting with antibodies against Tim23. Cross-linked dimers were quantified by densitometry. (B) Dependence of Tim23 dimer on respiratory chain substrate NADH. Mitochondria were preincubated for 15 min at 25°C in import buffer to deplete endogenous substrates. Subsequently NADH was added to the indicated concentrations and the mitochondria were incubated for 3 min before cross-linking was performed. One sample received 4 μM antimycin A (AA) together with 5 mM NADH. Crosslinks were visualized by SDS–PAGE and Western blotting (upper panel) and quantified by densitometry (lower panel). (C) A potassium diffusion potential supports formation of cross-linkable Tim23 dimer. Mitochondria were incubated in buffer containing 0.1 μM valinomycin, 12 μM oligomycin, and 4 μM antimycin A and then diluted into buffer containing the indicated concentrations of KCl. After 3 min at 25°C, DSG was added and dimers were determined by cross-linking. Upper panel, Western blot; lower panel, dimers quantified by densitometry. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 5 Tim23 Dimer Is Absent in Mitochondria with Preprotein Occupying the TIM Complex (A) Accumulation of chemical amounts of a preprotein in the TIM complex inhibits Tim23 dimer formation. Isolated mitochondria and mitoplasts from wild-type yeast overexpressing Tim23his12 were incubated with purified, radiolabelled pSu9(1–69)DHFR (280 ng preprotein, 200 μg mitochondria or mitoplasts). When indicated methotrexate (MTX) was included into the import reaction. After 3 min at 25°C, mitochondria and mitoplasts were reisolated and washed. Upper panel: aliquots (170 μg) of each sample were subjected to cross-linking with DSG and samples were analyzed by SDS–PAGE and Western blotting with antibodies against Tim23. Lower panel: aliquots of 30 μg were treated with 50 μg/ml proteinase K (+PK) and subjected to SDS–PAGE and analyzed by fluorography. p, i, m: precursor-, intermediate-, and mature-sized (respectively) forms of pSu9(1–69)DHFR. (B) The amount of Tim23 dimer in mitochondria is decreased by increasing amounts of preprotein. Energized mitochondria (200 μg) were incubated for 3 min at 25°C with the indicated amounts of pSu9(1–69)DHFR. Subsequently samples were placed on ice, cross-linking was performed and Tim23 dimer was analyzed. (C) Tim23 dimer is dissociated by upon import of preproteins and is reestablished after completion of import. Mitochondria were incubated in import buffer at 25°C. An aliquot (200 μg) was withdrawn for cross-linking before addition of preprotein (“Control”), after 3 min incubation with pSu9(1–69)DHFR (“Import”) and after reisolation and incubation of the mitochondria for 7 min at 25°C in fresh import buffer (“Chase”). Samples were analyzed by SDS–PAGE and immunoblotting with antibodies against Tim23. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 6 Tim23 Dimers Dissociate in a Presequence-Specific Manner (A) Dimers of the N-terminal domain of Tim23 dissociate upon interaction with the matrix targeting sequence of pSu9(1–69)DHFR. Tim23(1–101)GST and Tim23(51–101)GST (5 μg/ml) were incubated with purified pSu9(1–69)DHFR and DHFR (10 μg/ml). MTX was included to stabilize the folded DHFR domain. Then cross-linking with 10 μM DFDNB was performed. Analysis of cross-linked dimer was by SDS–PAGE and immunoblotting with antibodies against Tim23. m, monomer; d, dimer. (B) The N-terminal domain of Tim23 interacts with the presequence. Tim23(1–101) (2 μg/ml) was incubated with or without pF1β(1–32) prepeptide (5 μM). Cross-linking and analysis of the samples was performed as described under (A). arrowhead: cross-link of Tim23(1–101) with pF1β(1–32). Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)

Figure 7 A Putative Leucine Zipper Structure in the N-Terminal Domain of Tim23 (A) Secondary structure prediction of Tim23 and sequence alignment with leucine zipper motifs of various proteins. Upper part: Tim23 is schematically outlined. Predictions of α-helical regions (hatched boxes) and of the probability to form coiled-coils are shown. The programs SSP and Coils (window size of 14 residues) were used for the predictions (Lupas 1996). Lower part: sequence alignment of amino acid residues 61–81 of Tim23; hHSF1, human heat shock transcription factor 1 (Rabindran et al. 1993); Lac I, lac repressor (Friedman et al. 1995); hT3Rα1, human thyroid hormone receptor (Wagner et al. 1995); p53 (Jeffrey et al. 1995). (B) Hypothetical structure of a potential leucine zipper motif involved in Tim23 dimerization. Left part, top view of an antiparallel coiled-coil formed by dimerization of two helices (61–81) of Tim23 is depicted. Leucine side chains involved in dimerization (knobs on stalks) and negatively charged amino acid residues (−) are indicated. Right part, schematic side view of Tim23 dimer in the inner membrane (IM). The membrane integrated portion of Tim23 is hatched. Cell 1996 87, 33-41DOI: (10.1016/S0092-8674(00)81320-3)