Abstract
Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44.
Highlights
Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM)
Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex
An in Vivo Screen to Identify Inhibitors of Mitochondrial Protein Translocation—To identify modulators of the TOM/ TIM23 translocation system, we adapted the genetic screen that resulted in the identification of import components Tim44, Tim23, and Tim17, based on mislocalization of the Su9Ura3 fusion protein from mitochondria to the cytosol [14]; the targeting sequence, abbreviated Su9, is derived from subunit 9 of Neurospora crassa F1Fo ATPase and confers robust import
Summary
An in Vivo Screen to Identify Inhibitors of Mitochondrial Protein Translocation—To identify modulators of the TOM/ TIM23 translocation system, we adapted the genetic screen that resulted in the identification of import components Tim, Tim, and Tim, based on mislocalization of the Su9Ura fusion protein from mitochondria to the cytosol [14]; the targeting sequence, abbreviated Su9, is derived from subunit 9 of Neurospora crassa F1Fo ATPase and confers robust import. The strains grew at a similar rate when the medium was supplemented with uracil (Fig. 1C) These data confirm that the import defect in the tim mutant conferred growth in medium lacking uracil. For a positive control of cell growth, column 23 contained the tim23-2[Su9-URA3] strain with 1% DMSO. Of 25 candidates that were reconfirmed by repeating the screening assay, 6 compounds reproducibly increased the growth of the WT[Su9-URA3] strain (supplemental Table S2). Because the potential hits might nonspecifically damage mitochondria, we investigated mitochondrial membrane integrity and oxidative phosphorylation properties in the presence of the small molecules (Fig. 1, D–G). MB-10 incubation with purified mitochondria from HEK293T cells did not cause release of proteins as detected by Coomassie staining (Fig. 1E) or immunoblot analysis (Fig. 1F). Two compounds (designated MitoBloCK-10/MB-10 and MitoBloCK-11/MB-11) did not alter respiration. (Note that respiration for MB-10 is shown, and the rate of respiration is Ϫ0.55 nmol/s after MB-10 addition, which was similar to that in the presence of NADH (Fig. 1G).) The chemical name for MitoBloCK-10 is 3-fluoro-NЈ-
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