Fig. 1. Identification of a 56 kDa protein present in the TIM23 complex. (A) Purification of the TIM23 complex from N.crassa. Isolated mitochondrial membranes from a Neurospora strain, which harbors a Tim23 with an N-terminal His₉ tag, were solubilized in digitonin and passed over an Ni-NTA column. Bound material was eluted with an imidazole-containing buffer and applied to a Resource Q ion exchange column at low salt concentration. Elution was performed with a gradient of 10–500 mM NaCl. Fractions were analyzed by SDS–PAGE and silver staining. The TIM23 complex was eluted from the Resource Q column essentially in a single fraction (left panel). Coomassie Blue staining of such a fraction is also shown (right panel). The 56 kDa protein is indicated by arrowheads. (B) Alignment of the Neurospora 56 kDa protein with its homologs from S.cerevisiae (YPL063w), C.elegans (T25076) and H.sapiens (XM_053074). Partial and complete conservation of amino acid residues are indicated by shaded and black backgrounds, respectively. The predicted single transmembrane domain (TM) is underlined. The asterisk shows the predicted cleavage site by the mitochondrial processing peptidase for the Neurospora protein, and the double asterisk the determined N-terminal residue of the isolated protein.

Fig. 3. Specific association of Tim50 with the TIM23 complex. (A) Co-immunoprecipitation of Tim50 with TIM23 components. Wild-type Neurospora mitochondria were solubilized with digitonin and incubated with antibodies against Tim50, Tim23, Tim17 and Oxa1, or with pre-immune IgG pre-bound to protein A–Sepharose beads. The beads were collected by centrifugation, washed, and bound proteins were eluted with Laemmli buffer. The supernatants were diluted directly in Laemmli buffer. Pellets and supernatants were analyzed by SDS–PAGE and western blotting for the antigens indicated. ‘Total’ and ‘Supernatants’ represent 25% of material present in ‘Pellets’. (B) Co-purification of Tim50 with TIM23 complex and vice versa via Ni-NTA beads. Mitochondria were isolated from Neurospora strains in which either Tim50, Tim23, Tim22 or Oxa1 carried a His tag. They were solubilized with digitonin and incubated with Ni-NTA beads. After elution with imidazole-containing buffer, proteins were analyzed by SDS–PAGE and western blotting for the indicated mitochondrial components. T, total digitonin solubilized material, representing 25% of input; B, material bound to Ni-NTA.

Fig. 5. Mitochondria isolated from Tim50-depleted cells are defective in import of preproteins using the TIM23 translocase but not in import of preproteins using only the TOM complex or the TIM22 translocase. Mitochondria were isolated from wild-type cells (WT) and cells depleted of Tim50 for 33 h (Tim50↓). Radiolabeled precursor proteins were synthesized in reticulocyte lysate and incubated with the mitochondria for the indicated time periods at 25°C (A, B and E) or 12°C (C and D). The import reaction was stopped by placing samples on ice and adding 1 µM valinomycin. Samples were divided, and one-half was incubated with proteinase K (+PK). Reisolated mitochondria were subjected to SDS–PAGE and autoradiography (upper panel). PK-protected material was quantified (lower panel). (A) pSu9(1–86)DHFR, a fusion protein consisting of the N-terminal 86 residues of the presubunit 9 of ATP synthase and dihydrofolate reductase. (B) pF1β, the precursor of subunit β of ATP synthase. (C) pCoxVa, the precursor of cytochrome oxidase subunit Va. (D) pCoxVaΔTM, the pCoxVa precursor with deletion of the transmembrane domain. (E) CCHL, cytochrome c heme lyase; AAC, ADP/ATP carrier; Tim23Δ50, Tim23 with residues 1–50 deleted. The indicated precursors were imported for 15 min into WT and Tim50↓ mitochondria as described above. (F) Tim50-depleted cells accumulate Hsp60 precursor in vivo. WT and Tim50↓ cells were harvested at different time points after shift to glucose-containing medium. Total cell extracts were prepared by alkaline lysis and analyzed by SDS–PAGE and immunodecoration against Hsp60. p, precursor form of Hsp60; m, mature form of Hsp60.

Fig. 2. Subcellular and submitochondrial localization of the 56 kDa protein in Neurospora. (A) The 56 kDa protein is located in mitochondria. Neurospora cells were fractionated into a mitochondrial fraction (mitochondria), a post-mitochondrial pellet (PMP) and a soluble fraction (cytosol). The fractions were analyzed by SDS–PAGE and immunoblotting with antibodies against the 56 kDa protein, Tom40 and tubulin, as mitochondrial and cytosolic markers, respectively. (B) Mitochondria were titrated with digitonin to open successively the outer and inner membrane. Samples were treated with proteinase K (PK) and analyzed by SDS–PAGE and immunoblotting with antibodies against the 56 kDa protein, and Tom70, cytochrome c heme lyase (CCHL) and the mitochondrial processing peptidase (MPP) as markers for outer membrane, intermembrane space and matrix, respectively. (C) Alkaline extraction of the 56 kDa protein. Mitochondria were treated with 0.1 M sodium carbonate, and soluble (S) and membrane fractions (P) were separated by centrifugation and analyzed as in (A) using antibodies against an integral membrane protein, ADP/ATP carrier (AAC), and a soluble protein, MPP, as controls.

Fig. 4. Tim50 is essential for viability of yeast cells. (A) Disruption of the TIM50 gene. The TIM50 gene was disrupted in diploid cells by homologous recombination. Cells were sporulated and subjected to tetrad analysis. Only two out of four spores were viable in all tetrads analyzed, indicating that Tim50 is an essential protein in yeast. (B) Downregulation of Tim50 in cells affects their growth. Cells expressing Tim50 under the control of the GAL10 promoter (Gal-TIM50) and wild-type cells (WT) were grown in medium containing 0.1% galactose. At time point zero, they were shifted to medium containing 0.1% glucose; the cell number was set to 1. (C) Cells depleted of Tim50 (Tim50↓) show normal levels of other mitochondrial proteins. WT and Tim50↓ cells were harvested 33 h after shift to glucose. Mitochondria were isolated and analyzed by SDS–PAGE and western blotting for the indicated proteins.

Fig. 6. Cross-linking of Tim50 to preproteins in transit. (A) Precursor pb₂Δ19(167)DHFR_K5 was imported into isolated wild-type yeast mitochondria under various conditions and subjected to cross-linking with DFDNB as described in Materials and methods. Mitochondria were re-isolated and solubilized in SDS-containing buffer. Immunoprecipitation was performed with the antibodies indicated. The immunoprecipitates were analyzed by SDS–PAGE and autoradiography. PI, pre-immune serum. Top panel: import in the absence of a membrane potential ΔΨ and of methotrexate (MTX). The observed cross-linked products to Tim50 are marked with asterisks. Middle panel: import in the presence of ΔΨ and MTX. Lower panel: import in the presence of ΔΨ and absence of MTX. (B) The precursor of pb₂Δ19(167)DHFR_K5 was bound to mitochondria in the absence of a membrane potential. Mitochondria were re-isolated, re-energized and incubated further. At the time points indicated, aliquots were withdrawn for cross-linking or for SDS–PAGE directly. Upper panel: samples which were subjected to cross-linking and immunoprecipitation with antibodies against Tim50 (αTim50Npep) were analyzed as in (A). Lower panel: samples were subjected directly to SDS–PAGE, autoradiography and quantification. The amounts of bound precursor protein and imported mature protein at various time points were related to total precursor bound at time point zero in the absence of ΔΨ, which was set to 100%.