The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria

Abstract

The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence. AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region. An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein. We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein. The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex. AAC can cross the outer membrane with an internal segment first, i.e. in a loop formation. Each module of AAC is required for dimerization in the inner membrane. We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.

Fig. 1. Cross-linking of AAC preprotein on the mitochondrial surface yields multiple products with Tom70. (A) [³⁵S]AAC in reticulocyte lysate was incubated with yeast wild-type mitochondria in the absence of ATP at 25°C for 20 min. For sample 8, reticulocyte lysate containing unlabeled AAC–DHFR was also added. Mitochondria were re-isolated and, where indicated, treated with the cross-linking reagent MBS (1 mM) prior to immunoprecipitation with the indicated antibodies (under stringent conditions). The total cross-linked forms are shown in lane 1 (representing 15% of total used per immunoprecipitation). Lane 9 shows a lower exposure (10%) of lane 4. Tom70* indicates cross-links between AAC and Tom70. MSF_L and MSF_S are large and small subunits of MSF, respectively. (B) [³⁵S]AAC was incubated with wild-type (WT) or tomΔ mitochondria in the absence of ATP at 25°C for 20 min. Mitochondria were re-isolated and subjected to cross-linking using MBS prior to immunoprecipitation with anti-Tom70. Samples were subjected to SDS–PAGE and phosphoimage analysis. (C) Wild-type mitochondria were left untreated or cross-linked with MBS (1 mM) and subjected to SDS–PAGE and immunodecoration with anti-Tom70. Tom70₂, dimer of Tom70.

Fig. 2. Each module of AAC contributes to the yield of cross-linking to Tom70. (A) Schematic diagram of AAC constructs consisting of one or two of the three modules of AAC. (B) The [³⁵S]AAC constructs were bound to yeast wild-type mitochondria, cross-linked and immunoprecipitated with anti-Tom70 as described in the legend to Figure 1A. Tom70* and Tom40* are cross-links between AAC constructs and Tom70 or Tom40, respectively.

Fig. 3. Cooperation of AAC modules in the accumulation in the GIP complex. (A) Schematic diagram of the N- and/or C-terminal fusions of DHFR to AAC. The radiolabeled fusion constructs were incubated in the presence or absence of 20 µM MTX with mitochondria isolated from wild-type S.cerevisiae. Following incubation at 25°C for 20 min, samples were halved and one half was treated with proteinase K (Prot. K). Mitochondria were re-isolated and subjected to BN-PAGE and phosphoimage analysis. Western transfer of a BN-PAGE followed by immunodecoration with anti-Tom40 indicates the mobility of the 400 kDa complex (lane 1). (B) The radiolabeled AAC constructs were incubated with mitochondria at 25°C for 15 min in the presence of Δψ and in the absence or presence of ATP as indicated, followed by treatment with proteinase K and analysis by SDS–PAGE. (C) Schematic diagram of N- and C-terminal deletions of AAC fused to DHFR. The indicated radiolabeled constructs were imported in the presence of 20 µM MTX as described in (A). The samples were not treated with proteinase K.

Fig. 4. Translocation of AAC across the outer membrane can be initiated while both termini are still located on the cytosolic side. (A) The GIP-accumulated AAC fusion constructs are in proximity to Tom70 and Tom40. [³⁵S]AAC fusion proteins in reticulocyte lysate were incubated with isolated wild-type mitochondria in the presence of 20 µM MTX and subjected to cross-linking with MBS and immunoprecipitation. The total cross-linked forms are shown in lanes 1, 6 and 11 (5% of total used per immunoprecipitation). Tom70* and Tom40* are cross-links between the AAC construct and Tom70 or Tom40, respectively. The single asterisk indicates weak cross-links to Tom22/Tom20. (B) The GIP-arrested AAC fusion constructs can contact Tim10. Radiolabeled AAC–DHFR, DHFR–AAC or DHFR–AAC–DHFR was mixed with intact mitochondria (lanes 1, 2, 4–6, 8–10 and 12) or mitochondria that were pre-swollen to release intermembrane space content (lanes 3, 7 and 11) in the presence of MTX but in the absence of Δψ. Following incubation for 20 min at 25°C, mitochondria were re-isolated and some samples were subjected to swelling (lanes 4, 8 and 12). All samples were subjected to cross-linking with EGS (0.4 mM), followed by trypsin treatment where indicated (samples in lanes 2, 6 and 10) and immunoprecipitation with anti-Tim10 (all samples). Tim10*, cross-link between AAC construct and Tim10.

Fig. 5. Stepwise transfer of AAC modules from Tom70 to the GIP. (A) Radiolabeled AAC–DHFR, DHFR–AAC or DHFR–AAC–DHFR were incubated with wild-type mitochondria at 25°C for 20 min in the presence of 20 µM MTX but in the absence of ATP. Samples were halved and re-isolated mitochondria were resuspended in import buffer containing 20 µM MTX in the absence or presence of ATP and incubated at 25°C for 10 min. The mitochondria were re-isolated and incubated with MBS, followed by immunoprecipitation with anti-Tom70 and anti-Tom40. The samples were subjected to gradient SDS–PAGE and cross-linked bands were detected by phosphoimaging (indicated by asterisks). The total cross-linking patterns are shown in lanes 1, 4, 7, 10, 13 and 16 (representing 3% of the total used per immunoprecipitation). (B) [³⁵S]AAC was incubated with wild-type mitochondria in the absence of ATP (sample 1). The mitochondria were re-isolated and a chase reaction was performed (samples 2–5). The mitochondria were incubated by a stepwise increase of the temperature from 4°C [2.5 min (sample 2) to 10 min (sample 3)] to 12°C [an additional 2.5 min (sample 4)] and finally 25°C [10 min at each temperature (sample 5)] in import buffer containing ATP in the absence or presence of a Δψ. Cross-linking was performed with MBS and was analyzed by SDS–PAGE and autoradiography.

Fig. 6. Each module of AAC is required for assembly to the dimeric form in the inner membrane. The various [³⁵S]AAC constructs were incubated with wild-type mitochondria in the absence or presence of a Δψ. Samples were split and mitochondria from one half were swollen. All samples were subjected to proteinase K treatment prior to BN-PAGE and phosphoimage analysis.