Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role - PubMed (original) (raw)

Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role

T Krimmer et al. Mol Cell Biol. 2000 Aug.

Abstract

The import motor for preproteins that are targeted into the mitochondrial matrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the translocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and drives their translocation into the matrix. While different functional mechanisms are discussed for the mtHsp70-Tim44 machinery, little is known about the actual mode of interaction of both proteins. Here, we have addressed which of the three Hsp70 regions, the ATPase domain, the peptide binding domain, or the carboxy-terminal segment, are required for the interaction with Tim44. By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase domain interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70. The interaction of the ATPase domain with Tim44 is ATP sensitive. The peptide binding domain and carboxy-terminal segment are unable to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. We conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.

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Figures

FIG. 1

FIG. 1

Interaction of Tim44 and the ATPase domain of mtHsp70 (Ssc1) in the two-hybrid system. (A) Two-hybrid interaction of Ssc1(APC) and Ssc1(A) with Tim44. The reporter yeast strain Y190 was transformed with the two-hybrid plasmids expressing the indicated fusion proteins. Transformants were grown for 2 days at 30°C on minimal medium lacking tryptophan and leucine. One clone of each transformation was streaked out on minimal medium lacking tryptophan, leucine, and histidine but containing 3-aminotriazole and cultured for 3 days at 30°C. (B) The two-hybrid fusion proteins are expressed in yeast. The yeast strain Y190 was transformed as described in the legend to panel in A. One clone of each transformation was cultured overnight in minimal medium lacking tryptophane and leucine. Cells of a 1-ml liquid culture were resuspended in 15 μl of electrophoresis sample buffer and separated by SDS-PAGE. The proteins were detected by immunodecoration with antibodies directed against Hsp70 or Tim44. endog., endogenous (authentic) Hsp70s and Tim44 reacting with the antibodies.

FIG. 2

FIG. 2

Separation of mtHsp70 into domains and import into isolated mitochondria. (A) Ssc1 constructs used. (B) Import into mitochondria. Rabbit reticulocyte lysates with radiolabeled mitochondrial preproteins (2% [vol/vol] of import reaction mixture) were incubated with isolated yeast wild-type mitochondria (25 μg of mitochondrial protein/100 μl of import reaction mixture) for the indicated times in the presence (+) or absence (−) of a ΔΨ. Import was stopped by the addition of 1 μM valinomycin. Half of the samples were treated with proteinase K (Prot. K). After reisolation of the mitochondria and separation by SDS-PAGE, imported proteins were detected by digital autoradiography. The asterisk indicates a fragment of Ssc1(AP) that was generated in small amounts by proteinase K upon incubation of the construct with mitochondria in the presence of a ΔΨ, apparently representing an incompletely imported membrane-spanning form of the protein; the quantitations shown in the following figures did not include this fragment but are only based on the fully imported mature forms. m, mature form; p, precursor form.

FIG. 3

FIG. 3

ATP-sensitive interaction of the Ssc1 ATPase domain with Tim44. (A) Experimental approach. (B) ATP-sensitive coprecipitation with anti-Tim44. Rabbit reticulocyte lysate with radiolabeled mitochondrial preproteins (25% [vol/vol] of import reaction mixture) was incubated with isolated yeast mitochondria (50 μg of mitochondrial protein/100 μl of import reaction mixture) for 10 min in the presence of a ΔΨ. Import was stopped by the addition of 1 μM valinomycin, and the mitochondria were treated with proteinase K. A fraction of the import reaction mixture was taken as a control (lanes 1 and 4). The rest was split into halves and lysed in buffer containing Triton X-100 or digitonin in the absence or presence of ATP as indicated. Then, a coimmunoprecipitation with antibodies directed against Tim44 was performed. The proteins were separated by SDS-PAGE and detected by digital autoradiography. (C) Efficiency of coprecipitation. The amount of each Ssc1 construct coprecipitated with anti-Tim44 was quantified from 15 independent experiments. The coprecipitated amount of Ssc1(APC) under the Triton X-100 or digitonin condition was set to 100%, respectively. Error bars indicate the standard errors of the mean.

FIG. 4

FIG. 4

The coprecipitation of Ssc1 constructs with anti-Tim44 is specific. (A) Efficiency of coprecipitation of endogenous Ssc1 with anti-Tim44. Isolated yeast mitochondria were lysed with Triton X-100 and subjected to coprecipitation with antibodies directed against Tim44. Precipitated Ssc1 and Tim44 were identified by immunodecoration. A total of 5% of the nonprecipitated material is shown in sample 1. (B) Neither Ssc1 in reticulocyte lysate nor control preproteins in mitochondria are coprecipitated with anti-Tim44. Topmost two panels: the precursor form (p) and the mature-sized form (m) of Ssc1(APC) were synthesized and radiolabeled in reticulocyte lysates and subjected to coprecipitation with anti-Tim44 in the presence of Triton X-100. Bottom three panels: the precursors of Ssc1(APC), F1β-bla and Su9-F1β, were imported into mitochondria. The mitochondria were lysed with Triton X-100-containing buffer and subjected to coprecipitation with anti-Tim44 as described in the legend to Fig. 3B. A total of 2% of the material subjected to immunoprecipitation (reticulocyte lysate or mitochondria, respectively) is shown as a control (sample 1). (C) Control preproteins are not coprecipitated with anti-Tim44 under digitonin conditions. The experiment was performed as described for the bottom three images of panel B except that the mitochondria were lysed with digitonin instead of Triton X-100. (D) The Ssc1 constructs are stable upon lysis in the presence of ATP. Rabbit reticulocyte lysates containing the radiolabeled Ssc1 constructs were incubated with isolated mitochondria as described in the legend to Fig. 3B. After lysis in Triton X-100-containing buffer in the absence or presence of ATP, a precipitation with antibodies directed against Ssc1 was performed. The amount of precipitated proteins was determined by digital autoradiography, and the ratio of the signals in lane 2 to those in lane 1 is shown.

FIG. 5

FIG. 5

The association of Ssc1 constructs with Tim44 is not impaired in ssc1-2 mutant mitochondria. (A) Coprecipitation of Ssc1 constructs with anti-Tim44. The experiment was performed essentially as described in the legend to Fig. 3B with the following modifications. ssc1-2 mitochondria were used. The mutant phenotype was induced by incubation of the mitochondria for 15 min at 37°C as described in Materials and Methods. Triton X-100 or digitonin was used for lysis of the mitochondria as indicated. No difference in the association of Ssc1 constructs was observed when wild-type mitochondria were treated at 37°C or not (results not shown). (B) Control proteins are not coprecipitated with anti-Tim44 from ssc1-2 mitochondria. The experiment was performed as described in panel A except that the preproteins F1β-bla and Su9-F1β were included. (C) Association of Ssc1 constructs with Tim44 in wild-type and ssc1-2 mitochondria. The amounts of Ssc1 constructs coprecipitated with anti-Tim44 were determined from wild-type and ssc1-2 mitochondria, under both Triton X-100 and digitonin conditions (means of 15 independent experiments for each condition are shown, as described in the legends to Fig. 3 and 5A). Columns 1 to 3 and 5 to 7 show the ratios of coprecipitation of imported constructs between ssc1-2 and wild-type mitochondria. Columns 4 and 8 show the ratios of coprecipitation with anti-Tim44 for endogenous Ssc1-2 versus wild-type Ssc1 (determined by immunodecoration as described in Materials and Methods).

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