Daniel Jacobs

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40

Background: Nearly all nascent mitochondrial proteins are transported by the translocase of the outer membrane (TOM) complex. Results: The core Tom40 β-barrel domain exhibits four conductive levels and three distinct substrate binding affinities. Conclusion: Tom40 interactions with presequence substrates depend upon the channels conformation. Significance: Conformational rearrangements in Tom40 may regulate substrate interactions. Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

Significance Tubulin has emerged as a highly unexpected component of mitochondrial membranes involved in regulation of membrane permeability. This discovery has reawakened interest in the nature of the tubulin–membrane interaction to answer a new question: How does tubulin, a cytosolic protein famous for its role in microtubule structure and dynamics, come to target mitochondrial membranes? Here, using a combination of five biophysical methods, we study peripheral binding of tubulin to biomimetic membranes of different lipid compositions. We conclude that tubulin distinguishes between lamellar and nonlamellar lipids through a highly conserved amphipathic binding motif. Specifically, α-tubulin targets cell and organelle membranes by sensing lipid-packing defects via an amphipathic α-helix, with broad consequences for both normal cellular function and disease. Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.