title: Targeting and import mechanism of coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) into the mitochondrial intermembrane space.
abstract: Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX(9)C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys(193), and the active site cysteine in Mia40, Cys(55). Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.
authors.affiliation: Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0654, USA.
authors.forename: Manjula
authors.initials: M
authors.lastname: Darshi
authors.forename: Kristina N
authors.initials: KN
authors.lastname: Trinh
authors.forename: Anne N
authors.initials: AN
authors.lastname: Murphy
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D019943:Amino Acid Substitution; D003579:Cytochrome Reductases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology
chemical_list: C521110:CHCHD3 protein, human; C507282:CHCHD4 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors