Molecular characterization of the novel pathway for oxidation and assembly of mitochondrial inner membrane proteins

• Applicant: Dr. Dejana Mokranjac
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564271119

The biological function of the majority of proteins requires their translocation across at least one cellular membrane and subsequent assembly into multimeric protein complexes. In addition, the functions of many proteins depend on further modifications such as for example introduction of disulfide bonds. Despite years of extensive research, many steps of these processes and proteins involved still remain unknown. We have recently discovered a novel pathway for oxidation and assembly of mitochondrial inner membrane (IM) proteins and Dbi1 (disulfide bond formation in inner membrane proteins) as its first component. Dbi1 is a conserved, previously poorly characterized IM protein which interacts with the unassembled pool of Tim17, the central component of the presequence translocase in the IM, and is upregulated in cells with increased levels of unassembled Tim17. In the absence of Dbi1, the conformation of the presequence translocase is perturbed and the stability of Tim17 is reduced. In addition, through its conserved CxxC motif, Dbi1 is involved in formation of the disulfide bond in Tim17 in a manner independent of the disulfide relay system, the major oxidation-driven protein import pathway into mitochondria. Tim22, the central component of the carrier translocase, and Cox20, an assembly factor of the complex IV of the respiratory chain, are further substrates of Dbi1. The data available so far suggest that the substrates of this pathway are mitochondrial IM proteins with at least two transmembrane domains flanked by two cysteine residues that form a disulfide bond on the intermembrane space side of the IM. We proposed that Dbi1 recognizes its substrates through their specific cysteine residues, introduces the disulfide bond and then chaperones their further assembly into the respective complexes. Here, we aim at the molecular understanding of this pathway and will focus on the following questions. How is Dbi1 (re)oxidized to remain active as an oxidoreductase? Does it cooperate with additional proteins during oxidation and assembly of its substrates? How does Dbi1 function as a chaperone? What is the full spectrum of its substrates? What are the functional consequences of this pathway and when does it become critical for the cells? Is the presequence pathway redox regulated? To address these questions, we will combine biochemistry with cell biology, genetics and proteomics. We expect to gain important novel insights into the biogenesis and quality control of mitochondria.

• DFG Programme: Research Grants