1. Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.
- Author
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Muthukumar, Gayathri, Stevens, Taylor A., Inglis, Alison J., Esantsi, Theodore K., Saunders, Reuben A., Schulte, Fabian, Voorhees, Rebecca M., Guna, Alina, and Weissman, Jonathan S.
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MITOCHONDRIAL proteins , *MITOCHONDRIAL membranes , *MEMBRANE proteins , *MOLECULAR chaperones , *TOPOLOGY , *UBIQUINONES - Abstract
Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems. [Display omitted] • Genome-wide screens identify biogenesis factors for diverse ⍺-helical OMM proteins • Parallel pathways mediate cytosolic targeting based on substrate topology • The NAC complex selectively targets polytopic proteins to the outer membrane • TTC1 is a cytosolic chaperone involved in OMM signal-anchored protein biogenesis Muthukumar et al. use genome-wide and systematic arrayed CRISPRi screens to identify ⍺-helical OMM protein biogenesis and quality control factors that triage substrates by topology. They establish NAC as a targeting factor for polytopic proteins and identify TTC1 as a cytosolic chaperone for signal-anchored proteins, using a C-terminal hydrophobic groove. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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