Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44.
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.
From the Departments of Chemistry and Biochemistry and.
Non
N
Miyata
the Department of Chemistry, University of California Riverside, Riverside, California 92521.
Zhiye
Z
Tang
From the Departments of Chemistry and Biochemistry and.
Michael A
MA
Conti
From the Departments of Chemistry and Biochemistry and.
Meghan E
ME
Johnson
From the Departments of Chemistry and Biochemistry and.
Colin J
CJ
Douglas
From the Departments of Chemistry and Biochemistry and.
Samuel A
SA
Hasson
Microbiology, Immunology, and Molecular Genetics.
Robert
R
Damoiseaux
the Department of Chemistry, University of California Riverside, Riverside, California 92521.
Chia-En A
CA
Chang
From the Departments of Chemistry and Biochemistry and koehler@chem.ucla.edu.|the Molecular Biology Institute, and.|the Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California 90095 and.
Carla M
CM
Koehler
D000818:Animals; D001665:Binding Sites; D005820:Genetic Testing; D006367:HeLa Cells; D006801:Humans; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009492:Neurospora crassa; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish
D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; C077016:TIM44 protein, S cerevisiae
chemical biology; mitochondria; mitochondrial transport; protein translocation; small molecule