Radhakrishnan MAHALAKSHMI

Indian Institute of Science Education and Research (IISER), Bhopal, INDIA

Correlating Assembly Mechanisms with Functional Regulation of the Human Mitochondrial Protein Import Channel

Host: N. Bhavesh

Nearly all mitochondrial proteins are generated cytosolically, and imported to various mitochondrial compartments by the Translocase complex of the Outer Mitochondrial Membrane (TOM). The core import protein, Tom40, is a structurally unique 19–stranded transmembrane β-barrel that hetero-oligomerizes with the smaller TOM subunits to form the functional TOM complex in the outer mitochondrial membrane (OMM). Tom40 is indispensable for mitochondrial biogenesis and mitostasis. Structural and functional studies with yeast TOM, combined with dysfunctional protein variants identified in various neurodegenerative diseases and cancers, have provided basic insights on how Tom40 is organized in the OMM, and has allowed for potential substrate import pathways to be identified. Yet, our limited understanding of the structural energetics, function, and regulation of this essential protein has hindered its use as therapeutics for mitochondrial disorders. For example, Tom40 is implicated in the misimport of α-synuclein, and the onset of neurodegenerative states. Understanding Tom40 assembly and identifying its molecular regulators will facilitate the discovery of drugs that target and modulate its function in vivo. Here, we combine ultrafast measurements of human Tom40 folding pathways, with single molecule studies of its function. Additionally, we identify thermodynamic elements that facilitate the correct assembly of this protein in membrana. We find that Tom40 assembles through parallel pathways with structured transition states that show folded C-terminal β-strands and an unfolded N-terminal region. Our proposed assembly mechanism correlates surprisingly well with the chaperone–assisted in vivo folding, revealing that Tom40 β-barrel folding pathway is coded in its primary sequence, and that the membrane chaperones serve merely as guides for directed C-to-N β-barrel folding. The C-terminal strands additionally serve as post-folding anchors for Tom40, and correlate well with interactomes mapped in vivo with the smaller TOM subunits. We also identify that residues with thermodynamically lowered stability are retained for Tom40 function and β-barrel malleability in vivo for efficient substrate import. Our findings provide the first folding–stability–function correlation of this essential human channel, opening avenues for identifying Tom40 target sites for designed peptidomimetics that bind and modulate Tom40 function and prevent α-synuclein misimport.


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