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Identification and functional analysis of human Tom22 for protein import into mitochondria - PubMed

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Comparative Study

Identification and functional analysis of human Tom22 for protein import into mitochondria

M Yano et al. Mol Cell Biol. 2000 Oct.

Abstract

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.

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Figures

FIG. 1
FIG. 1

Comparison between human and fungal Tom22 sequences. Amino acids are designated with the single-letter code, and gaps were introduced to maximize the alignment. The identical or similar residues (I/V/L, R/K, G/A, D/E, Q/N, T/S, and F/Y) are shown in shaded blocks. Putative transmembrane regions are boxed. S.c., S. cerevisiae; N.c., N. crassa.

FIG. 2
FIG. 2

Localization of mammalian Tom22. (A) COS-7 cells were harvested and suspended in hypotonic buffer. After sonication, the suspension was centrifuged at 500 × g and the supernatant was used as whole-cell extract. The cell extract was further centrifuged at 100,000 × g to give the soluble and membrane fractions. The membrane fraction was extracted with alkali (0.1 M Na2CO3 [pH 11.5]). The whole-cell extracts (W) (44 μg of protein), soluble fractions (S) (23 μg), membrane fractions (M) (24 μg), precipitated fraction (AP) (11 μg), and soluble fraction (AS) (12 μg) after extraction with alkali were subjected to SDS–12% PAGE and immunoblot analysis using affinity-purified antibodies to the N-terminal portion (anti-T22N) and the C-terminal portion (anti-T22C) of hTom22. Mitochondrial porin (outer membrane protein) and Hsp60 (matrix protein) were also stained as controls. (B) Rabbit reticulocyte lysate (10 μl) containing 35S-labeled hTom22 was incubated with isolated mitochondria (50 μg of protein) for 40 min at 25°C. Mitochondria were precipitated and subjected to extraction with alkali. The precipitated mitochondria (P) and the alkali-insoluble fraction (AP) were subjected to SDS–14% PAGE. “20%” represents 20% of hTom22 input in the import assay. (C) COS-7 cells grown on coverslips in 35-mm-diameter culture dishes were transfected with 2 μg of pCAGGS-pOTC-GFP. After culture for 24 h, cells were subjected to immunostaining with anti-T22C antibody and secondary antibody labeled with Cy3. Fluorescence due to Cy3 (a and c) or GFP (b and d) was photographed. Arrowheads indicate the cells expressing pOTC-GFP. (D) Isolated mitochondria (20 μg) from COS-7 cells were treated with trypsin (200 μg/ml) or proteinase K (PK) (200 μg/ml) in the absence or presence of 0.5% Triton X-100 for 30 min on ice. The products were subjected to Tris-Tricine PAGE, followed by immunoblot analysis with anti-T22C or anti-T22N antibodies. Numbers at left are molecular masses in kilodaltons.

FIG. 3
FIG. 3

Effect of anti-hTom22 antibodies on import of preproteins into isolated mitochondria. (A) Isolated COS-7 cell mitochondria (25 μg) were preincubated in the presence of 6 μg of nonimmune IgG (NI) or affinity-purified anti-T22N antibody (22N) for 20 min at 25°C in the import reaction mixture (50 μl). 35S-labeled reticulocyte lysate translation product containing human pOTC (10 μl) was then added to the mixture and incubated at 25°C. The import reaction was stopped at the indicated times and subjected to SDS–10% PAGE. The radioactive polypeptides were visualized by image plate analysis (left panels), and the radioactive mature OTC on the SDS-polyacrylamide gels was quantified (right panel). “20%” represents 20% of input pOTC. The percent import represents the amount of mature OTC compared with the input pOTC. (B) Import of 35S-labeled human pOTC was performed in the absence (control) or presence of indicated amounts of nonimmune IgG (NI) or affinity-purified anti-T22N (22N) or anti-T22C (22C) antibodies for 16 min at 25°C and was analyzed as described for panel A. Import without antibody was set as 100%. (C) Import of human pOTC (hpOTC), pOTC-GFP, rat pOTC (rpOTC), pre-serine:pyruvate aminotransferase (pSPT), and pre-aspartate aminotransferase (pAAT) was performed in the presence of 6 μg of nonimmune IgG (NI) or affinity-purified anti-T22N antibody (22N) for 16 min at 25°C and was analyzed as described for panel A. The percent import represents the amount of mature proteins compared with the input precursors. Values are represented by means ± standard deviations of three independent experiments. p and m, precursor and mature forms, respectively.

FIG. 4
FIG. 4

Interaction between Tom22 and Tom20. (A) COS-7 cells were lysed in 10 mM MOPS-NaOH (pH 7.2) containing 0.5% digitonin, 250 mM sucrose, 1 mM EDTA, 200 mM NaCl, and 3% bovine serum albumin. The cell lysates were centrifuged at 25,000 × g for 15 min at 4°C, and the soluble fractions (340 μg of protein) were subjected to immunoprecipitation using anti-Tom20, anti-hT22C, or anti-hT22N antiserum or preimmune serum as described in Materials and Methods. The immunoprecipitates were subjected to SDS–12% PAGE and immunoblot analysis. W, COS-7 cell extracts (38 μg of protein). (B) COS-7 cells were transfected with 10 μg of pCAGGS (control) or pCAGGS-hTom22. After culture for 20 h, cells were harvested and fractionated as described in Materials and Methods. Whole-cell extracts (W) (30 μg), soluble fractions (S) (18 μg), and membrane fractions (M) (15 μg) were subjected to SDS–12% PAGE and immunoblot analysis for the indicated proteins.

FIG. 5
FIG. 5

Cooperation of hTom22 and hTom20 on preprotein import. COS-7 cells were cultured in 10-cm-diameter dishes. (A) Two micrograms of pCAGGS-pOTC was cotransfected with 4 μg of pCAGGS (control), pCAGGS-hTom20, or pCAGGS-hTom22. After culture for 20 h, cells were harvested, and cell extracts (45 μg of protein) were subjected to SDS–12% PAGE and immunoblot analysis for the indicated proteins. (B) Two micrograms of pCAGGS-pOTC was cotransfected with indicated amounts of plasmids. After 16 h of culture, cells were harvested and subjected to pulse (5-min)-chase (40-min) experiments as described in Materials and Methods. The radioactive pOTC and mature OTC (mOTC) on the SDS-polyacrylamide gel were quantified by image plate analysis, and percentages of mOTC versus pOTC plus mOTC are shown. Values are represented by means ± standard deviations of three independent experiments. p and m, precursor and mature forms, respectively.

FIG. 6
FIG. 6

Effect of GST-fused hTom22s on mitochondrial import of pOTC, pOTCN-GFP, and pOTC-GFP. (A) GST or GST-fused hTom22s and Tom20 were expressed and purified as described in Materials and Methods. Purified proteins (5 μg) were subjected to SDS–10% PAGE. T20, GST-(25–145)hTom20; T22C, GST-(102–142)hTom22; 48, GST-(1–48)hTom22; 62, GST-(1–62) hTom22; 71, GST-(1–71)hTom22; 75, GST-(1–75)hTom22; 79, GST-(1–79) hTom22; 82, GST-(1–82)hTom22. (B) Import of 35S-labeled human pOTC translated in reticulocyte lysate (10 μl) into isolated mitochondria (25 μg) was performed in the absence (control) or presence of indicated amounts of purified GST or GST-fused hTom22 derivatives at 25°C for 16 min in the import reaction mixture (50 μl) and was analyzed as described for Fig. 3. Ten percent of input preprotein was placed in the first lane. (C) Mitochondrial import of 35S-labeled human pOTC, pOTCN-GFP, and pOTC-GFP was performed in the presence of 1 nmol of GST or GST-fused hTom22 derivatives at 25°C. At the indicated times, the import reaction was stopped and analyzed as described for Fig. 3. (D) Mitochondrial import was performed in the presence of 1 nmol of GST (control), GST-fused hTom22 derivatives, and GST-fused hTom20 at 25°C for 16 min for human pOTC, 20 min for pOTCN-GFP, or 40 min for pOTC-GFP. The import reaction was stopped and analyzed as described for Fig. 3. Import was expressed as percentage of controls in which GST was included. Values are represented by means ± standard deviations of three independent experiments. Solid bars, pOTC; shaded bars, pOTCN-GFP; open bars; pOTC-GFP. p and m, precursor and mature forms, respectively.

FIG. 7
FIG. 7

Preprotein binding to hTom22 and hTom20. (A) 35S-labeled translation product (10 μl) was incubated for 30 min at 25°C with glutathione-agarose prebound with about 0.56 nmol of GST-(1–82)hTom22 or GST-(25–145)hTom20. After washing, GST derivatives were eluted with 15 mM reduced glutathione, and 40% of the eluted protein was subjected to SDS–10% PAGE and fluorography using a FUJIX BAS2000 analyzer as described in Materials and Methods. Five percent of input preproteins were put in the first lanes. R23A, R23A pOTC-GFP; R15/23/26A, R15/23/26A pOTC-GFP. (B) The radioactive preproteins eluted were quantified by image plate analysis. The binding was expressed as percentage of input precursors. Values are represented as means ± standard deviations of three independent experiments. (C) The binding assay was performed as described for panel A except that the indicated concentrations of KCl or Triton X-100 were added to binding and washing buffers. Binding was expressed as percentage of controls without KCl and Triton X-100.

FIG. 8
FIG. 8

Analysis of sites of interaction of hTom22 with hTom20. Purified GST-fused hTom22s (each 0.2 nmol) were incubated with the same amount of GST-(25–145)hTom20 (T20) for 30 min at 25°C in the lysis buffer used for the coimmunoprecipitation assay. After centrifugation, immunoprecipitation using anti-Tom20 antiserum was performed as described in Materials and Methods. Ten percent of the immunoprecipitated proteins and 10% of the input proteins were subjected to SDS–12% PAGE and immunoblot analysis using an anti-GST antibody conjugated with horseradish peroxidase. Asterisks indicate the coprecipitated Tom22 proteins.

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