Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation

Abstract

The TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical-genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.

Fig. 1.

Phenotypic analysis of the strains used for the chemical synthetic-lethality screen for inhibitors of the TIM22 protein import pathway. (A) Growth phenotypes of the control (TIM10), the tim10-1 mutant, and tim10-1 suppressor (tim10-1 tim9S) strains used in the screen. Strains were plated on rich glucose (YPD) or ethanol-glycerol (YPEG) media and incubated at 25 °C or 37 °C. All of these strains were isogenic except for their denoted genetic variation. (B) Radiolabeled AAC was imported into isolated mitochondria in the presence and absence of a membrane potential (Δψ). Aliquots were removed at the indicated time points and samples were treated with carbonate extraction to confirm that AAC was inserted into the IM.

Fig. 2.

MitoBloCK-1 exhibits a chemical synthetic lethality with the tim10-1 mutant. (A) The structure of MitoBloCK-1, a tetrahydrodibenzofuran compound. (B) MIC₅₀ analysis of two tim10 mutants (tim10-1 and tim10-73) and the parental (TIM10) strain with MitoBloCK-1. Average%survival ± SD of n = 3 trials. The R² value for tim10-1 and tim10-73 curve fits were 0.98 and 0.99, respectively.

Fig. 3.

MitoBloCK-1 inhibits the import of substrates that use the TIM22 import pathway. Import assays were performed with radiolabeled precursors into mitochondria from the tim10-1 tim9S suppressor strain, which has restored import of AAC. Time course assays were completed with various concentrations of MitoBloCK-1 or the vehicle control (1% DMSO). Nonimported precursor was removed by protease treatment. Precursors include (A) AAC, (B) the phosphate carrier (PIC), (C) Tom40, and (D) Hsp60, (A–C) represent precursors that use the TIM22 import pathway whereas D is a substrate of the TIM23 import pathway. p, precursor; m, mature.

Fig. 4.

MitoBloCK-1 impairs substrate binding by the Tim9-Tim10 complex. (A) AAC was imported into mitochondria isolated from TIM10, tim10-1, and suppressor tim10-1 tim9S strains in the presence and absence of a Δψ. Where indicated, MitoBloCK-1 was included in the tim10-1 tim9S mitochondria. After importing AAC 15 min, reactions were stopped with either cold buffer or trypsin (protease). (B) AAC was imported into tim10-1 tim9S mitochondria in the presence of 25 μM MitoBloCK-1 or uncoupled mitochondria (lanes 1–3), A fraction of the import reaction was treated with the irreversible cysteine cross-linker bismaleimidohexane (BMH) (lanes 4–6). BMH-treated samples were divided and aliquots were subjected to immunoprecipitation (IP) with either Tim22 (22), Tom40 (40), or Tim9 (9) polyclonal antibodies bound to protein A-Sepharose beads (lanes 7–12). In addition to the previously characterized Tim9-AAC cross-link, a second cross-link of approximately 55 kD (denoted by *) was prevalent in the MitoBloCK-1 and BMH treated sample (lane 6).

Fig. 5.

MitoBloCK-1 facilitates substrate specificity analysis. Tim22 (A), Tim23 (B), and Tafazzin (C) were imported into tim10-1 tim9S mitochondria in the presence of MitoBloCK-1 or the vehicle (1% DMSO) followed by carbonate extraction to confirm insertion into the membrane.

Fig. 6.

MitoBloCK-1 activity is influenced by specific chemical characteristics and inhibits AAC imported into mammalian mitochondria. (A) Analogs of MitobloCK-1 were purchased from Chembridge and assayed in import assays with radiolabled AAC as previously described. (B) AAC was imported into isolated mouse liver mitochondria in the presence of 25 μM MitoBloCK-1 as in Fig. 3A. (C) Model of MitoBloCK-1 activity from experimental evidence. See text for more details.