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Functional Assembly of the Mitochondrial Protein Transport Machinery
Author(s) -
Alder Nathan,
Peixoto Pablo,
Modak Arnab
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00154
Subject(s) - microbiology and biotechnology , protein targeting , mitochondrial membrane transport protein , translocase of the inner membrane , protein subunit , biology , biogenesis , transport protein , mitochondrial carrier , membrane protein , mitochondrion , biochemistry , inner mitochondrial membrane , bacterial outer membrane , membrane , escherichia coli , gene
The vast majority of proteins that reside within mitochondria are encoded in nuclear DNA, synthesized on cytosolic ribosomes, and imported into the organelle. Within the mitochondrion reside several types of complex machineries that mediate the sorting, translocation and integration of incoming polypeptides based on their targeting information. Among these translocases, the TIM23 Complex mediates the import and biogenesis of most resident proteins, which are synthesized with cleavable amino‐terminal targeting sequences. The TIM23 Complex is a dynamic multi‐subunit assembly that serves versatile functions in mediating the transport of soluble proteins across the mitochondrial inner membrane as well as the lateral integration of membrane proteins. The multifunctional nature of this complex may arise from different modular states that it can assume based on its component subunits. Using a host of biophysical approaches, we have investigated the substrate‐ and energy‐dependent structural dynamics of the central subunit, the voltage‐gated channel Tim23. We have also analyzed the lipid interactions that mediate membrane associations with the receptor subunit, Tim50, as well as channel‐receptor interactions. To investigate the structure and function of the membrane‐sorting modular form of TIM23, we have utilized a reductionist approach in which five component subunits are purified from a heterologous expression system and reconstituted into a biomimetic model membrane. These subunits include the polytopic channel proteins Tim23 and Tim17, the Tim50 receptor, and additional membrane proteins Tim21 and Mgr2. The innovation of this system derives from the fact that multiple independently purified membrane proteins can form a functional complex spontaneously when reconstituted with the proper topology in a bilayer of synthetic lipids. This reconstituted complex provides a minimal system for the direct analysis of its channel features, its energetic requirements for mediating membrane protein integration, and the factors that mediate assembly, including lipid requirements. It also provides a highly purified system for structural analysis. Support or Funding Information This work was supported by NIH Grant 1R01GM113092 (to N.N.A.) and PSC‐CUNY Grant PSC‐CUNY 626860050 (to P.P.)