title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles (UCLA), Los Angeles, California, USA.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Hasson
authors.forename: Robert
authors.initials: R
authors.lastname: Damoiseaux
authors.forename: Jenny D
authors.initials: JD
authors.lastname: Glavin
authors.forename: Deepa V
authors.initials: DV
authors.lastname: Dabir
authors.forename: Scott S
authors.initials: SS
authors.lastname: Walker
authors.forename: Carla M
authors.initials: CM
authors.lastname: Koehler
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D006835:Hydrazones / Q000737:chemistry / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D015394:Molecular Structure; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity; D013890:Thiourea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
chemical_list: D006835:Hydrazones; D008565:Membrane Proteins; C550341:MitoBloCK-1; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; C107250:TIM10 protein, S cerevisiae; C102549:TIM22 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae; D013890:Thiourea
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