Tubulin on Biomimetic Mitochondrial Membranes: Structural Features and Lipid Discrimination

Abstract

Dimeric tubulin, an abundant cytosolic water-soluble protein, has emerged as an important regulatory factor of the permeability of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. A wealth of recent in vitro and in vivo evidence points to a mechanism of this regulation, in which a charged C-terminal tail of tubulin inserts into the water-filled VDAC pore, thus blocking metabolite fluxes through this passive transport channel. The membrane-binding properties of tubulin have proven difficult to ascertain, as early work generated contradictory results regarding the tubulin-membrane association, including whether it was integral or peripheral in nature. Here we report on a comprehensive biophysical study of tubulin binding to lipid membranes with compositions that mimic the mitochondrial outer membrane. A combination of surface plasmon resonance, bilayer overtone (second harmonics) analysis, and single-channel recordings show that tubulin distinguishes between lamellar and non-lamellar lipid components of the membrane. To obtain the structural features of the tubulin heterodimer on the membrane surface, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane platform. The NR results definitively show that tubulin binds peripherally, and in combination with molecular dynamics (MD) simulations suggest the binding domain to be a highly conserved amphipathic α-helix on α-tubulin. Thus tubulin joins a short but growing list of amphitropic proteins that target cell and organelle membranes by sensing lipid packing defects via amphipathic α-helices, suggesting a pathway by which lipid homeostasis regulates mitochondrial function.