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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02526

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Import of rat mitochondrial citrate carrier (CIC) at increasing salt concentrations promotes presequence binding to import receptor Tom20 and inhibits membrane translocation

¹ Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università di Lecce, I-73100 Lecce, Italy
² Institut für Mikrobiologie, Universität Hohenheim, 70593 Hohenheim, Germany
³ Dipartimento Farmaco-Biologico, Università di Bari, I-70125 Bari, Italy

View larger version (34K): [in a new window]   Fig. 1. Import of CIC into isolated rat liver mitochondria. (A) Isolated rat liver mitochondria were preincubated with antibodies directed against Tom20 or Tom70, respectively, at increasing concentrations. Reticulocyte lysate containing the 35S-labeled pCIC or mCIC was added and the samples were incubated for 20 minutes at 25°C. The mitochondria were then cooled-down to 0°C and treated with 50 µg/ml proteinase K to remove non-imported protein. Proteolysis was stopped by addition of PMSF, the mitochondria were isolated again by centrifugation, and imported proteins were analyzed by SDS-PAGE and autoradiography. The relative amounts of 35S-labeled protein were determined using a phosphoimager. The amounts of imported protein in the absence of antibodies were set to 100% (control). (B) Import of AAC and Su9-DHFR (a hybrid protein containing the presequence of subunit 9 of the mitochondrial ATP synthase coupled to dihydrofolate reductase), following the same protocol as in the experiments of A. p, precursor protein; m, mature protein.

View larger version (24K): [in a new window]   Fig. 2. Import of CIC into isolated yeast mitochondria. (A) Mitochondria were isolated from yeast strains lacking the import receptor Tom70 (tom70) or Tom20 (tom20). Wild-type mitochondria (WT) were isolated in parallel. 35S-labeled pCIC was synthesized in reticulocyte lysate and incubated with the isolated mitochondria at 25°C. Aliquots were removed at different time points as indicated and cooled-down to 0°C. The mitochondria were incubated with 250 µg/ml proteinase K for 20 minutes and isolated again by centrifugation. Imported proteins were analyzed by SDS-PAGE and fluorography. The relative amount of imported pCIC was determined using a phosphoimager. The highest value was set to 100% (control). Within import-times of 10 minutes, up to 11% of added pCIC were imported. (B) Import of mCIC, following the same protocol as described in A. (C) Import of Su9-DHFR. (D) Import of AAC.

View larger version (21K): [in a new window]   Fig. 3. Import of CIC into mitochondria that lack components of the TOM complex. (A) 35S-labeled pCIC and mCIC were synthesized in reticulocyte lysate and incubated for 10 minutes at 25°C with isolated yeast mitochondria lacking Tom5. Mitochondria of the corresponding wild type were used in parallel assays. The import reaction was stopped by treatment with 250 µg/ml proteinase K at 0°C. The mitochondria were isolated again and the mitochondrial proteins were separated by SDS-PAGE. The relative amounts of imported CIC were determined using a phosphoimager. (B) Import of 35S-labeled pCIC and mCIC into mitochondria that lack Tom22, following the same protocol as described in (A). (C) Import of 35S-labeled Su9-DHFR into isolated mitochondria of the yeast strain tom22-2. (D) Import of pCIC into tom22-2 mitochondria.

View larger version (20K): [in a new window]   Fig. 4. Import of pre-proteins into mitoplasts. (A) Mitoplasts were prepared by osmotic shock (swelling, SW) of yeast mitochondria. Reticulocyte lysate containing 35S-labeled Su9-DHFR was incubated for 20 minutes at 25°C with mitochondria (lanes 1 and 2) or mitoplasts (lanes 3 and 4). Valinomycin and oligomycin were added to samples 2 and 4 to dissipate the membrane potential (-). To degrade non-imported protein, 50 µg/ml proteinase K were subsequently added to all samples. The mitochondria were isolated again and analyzed by SDS-PAGE and fluorography. (B) Import of AAC, pCIC and mCIC. The experiment was performed as described in A. (C) Import of AAC, pCIC, and mCIC into intact mitochondria. The 35S-labeled proteins were imported into intact yeast mitochondria for 20 minutes at 25°C. The mitochondria of samples 3 and 4 were subsequently subjected to osmotic shock (+ SW). Following treatment with proteinase K, mitochondria and mitoplasts were isolated again by centrifugation.

View larger version (23K): [in a new window]   Fig. 5. Participation of Tim44 and mitochondrial Hsp70 (mtHsp70) in import of pre-proteins. (A) Mitochondria were isolated from the temperature-sensitive yeast strain tim44-8, wild-type mitochondria (WT) were isolated in parallel under the same conditions. To induce the phenotype, the isolated mitochondria were incubated for 15 minutes at 37°C. The mitochondria were then incubated with reticulocyte lysate containing 35S-labeled pCIC at 25°C. Samples were taken after different time points as indicated, and treated with 250 µg/ml proteinase K for 20 min at 0°C. The proteins were subjected to SDS-PAGE and autoradiography. The relative amounts of imported pCIC were determined using a phosphoimager. The highest value was set to 100% (control). (B) Import of Su9-DHFR. The experiment followed the same protocol as described in A, using mitochondria of the same preparation. (C) Import of AAC. (D) Import of pCIC into mitochondria of the temperature-sensitive yeast strain ssc1-3. The gene SSC1 encodes the major 70 kDa heat shock protein of the mitochondrial matrix. (E) Import of Su9-DHFR into mitochondria from the same preparation as in experiment D. (F) Import of AAC.

View larger version (30K): [in a new window]   Fig. 6. Inhibition of protein import by increasing concentrations of KCl. (A) AAC and Su9-DHFR were synthesized in reticulocyte lysate in the presence of 35S-labeled methionine. The reticulocyte lysates were diluted with BSA-buffer pH 7.2, containing different concentrations of KCl as indicated. Wild-type yeast mitochondria were added and the mixtures were incubated for 10 minutes at 25°C. The samples were then cooled-down to 0°C and proteinase K was added to digest non-imported protein. The mitochondria were isolated again by centrifugation and analyzed by SDS-PAGE and autoradiography. The relative amounts of imported protein were determined using a phosphoimager. The amounts of AAC or Su9-DHFR that had been imported in the presence of 80 mM KCl were set to 100% (control). (B) Import of pCIC and mCIC, following the same procedure as in A. (C) Import of pCIC into wild-type mitochondria (WT), and into mitochondria lacking the import receptor Tom70 (tom70) or Tom20 (tom20), respectively. The import experiment essentially followed the scheme described in A, except that the samples contained either 80 mM KCl (-) or 400 mM KCl (+). (D) Import of mCIC, following the same protocol as described in C. (E) Import of pCIC into wild-type mitochondria. The mitochondria were pretreated with 100 µg/ml trypsin for 15 minutes at 0°C (+) or left without trypsin (-). The mitochondria were incubated with pCIC for 10 minutes at 25°C. The samples were subsequently treated with 250 µg/ml proteinase K (+ PK) or left without protease (- PK). The amounts of imported pCIC were determined as described in (A). (F) Relative amounts of imported pCIC vs bound pCIC after import in the presence of different KCl concentrations. The experiment was carried out as described in B, using parallel samples +/- treatment with proteinase K.

View larger version (30K): [in a new window]   Fig. 7. Inhibition of protein import into both rat liver and yeast mitochondria by high ionic strength. (A) AAC and Su9-DHFR were synthesized in reticulocyte lysate in the presence of 35S-methionine. Isolated rat liver mitochondria were added in the presence of increasing concentrations of KCl as indicated. The samples were incubated at 25°C for 20 minutes, cooled-down to 0°C and treated with proteinase K. The mitochondria were isolated again and analyzed by SDS-PAGE and fluorography. The relative amounts of imported protein were calculated, the values determined for the 80 mM KCl sample were set to 100% (control). (B) Import of pCIC and mCIC, following the same procedure as in A. (C) Precursor (pPiC) and mature phosphate carrier lacking the presequence (mPiC) were synthesized in reticulocyte lysate in the presence of 35S-methionine. Isolated yeast mitochondria (wild-type) were added in the presence of increasing concentrations of KCl as indicated. The samples were then treated as described in (A). (D) pPiC and mPiC were imported into isolated yeast mitochondria as described in C. Standard samples of the reticulocyte lysate were included in the SDS-PAGE to determine the ratio of imported PiC vs the total amount of PiC added to each sample.

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