Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity

Abstract

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. While freely diffusing TOM-CC molecules are predominantly in a high permeability state, non-mobile molecules are mostly in an intermediate or low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus represent a β-barrel membrane protein complex to exhibit membrane state-dependent mechanosensitive properties, mediated by its two Tom22 subunits.

New insights into the structure and dynamics of the TOM complex in mitochondria

“…Biochemical [ 73 ], electrophysiological [ 74–77 ] and recent structural data of the TOM holo complex with the peripherally associated subunit Tom20 [ 14 , 16 ] suggested different structural states indicating significant dynamic conformational changes of the TOM complex. It was, therefore, surprising that mechanical interactions of the Tom22 preprotein receptors with structures in the vicinity of the membrane lead to conformational changes that result in the reversible closure of the two Tom40 pores ( Figure 4 ) [ 19 ]. Freely diffusing TOM core complex molecules were found to ‘stall’ when interacting with structures adjacent to the membrane, presumably due to the local confinement of its extended polar IMS domains of Tom22.…”

“… ( A ) Experimental TIRF setup for tracking individual TOMs and imaging their ion channel activities [ 18 , 19 ]. Membranes are formed by contact between aqueous droplets coated with a lipid monolayer in a lipid/oil phase and a lipid monolayer on an agarose hydrogel.…”

“…( B ) Fluorescence signals reveal the local position of individual TOMs, which is used to determine their mode of lateral mobility (lateral movement vs. transient anchoring) in the membrane and the corresponding permeability states of the molecule, indicated by the level of fluorescence (low, medium and high). ( C ) Trajectories and open-closed activities of a single TOM core complex [ 18 , 19 , 88 ]. A lateral stall (red) of freely moving (blue) TOM molecules is accompanied by a partial or complete closure of a Tom40 channel.…”

“…Subsequently, we shift our focus to the present state of understanding regarding the channel dynamics observed in vitro . Employing cutting-edge experimental techniques involving supported droplet interface lipid bilayers and single-molecule total internal reflection fluorescence (TIRF) microscopy [ 17 , 18 ], recent studies have demonstrated that tethering TOM to structures adjacent to the membrane induces the closure of its two protein translocation pores [ 19 ]. In view of the fact that structural data indicate a direct interaction of TOM with the IMM protein translocase TIM23 during the translocation of proteins into the mitochondrial matrix [ 20–23 ], it is tempting to infer a functional role for the in vitro mechanosensitivity of TOM channel activity from the nature of the functional interactions with the proteins of the inter membrane space (IMS) and IMM.…”

Two conformations of the Tom20 preprotein receptor in the TOM holo complex

“…Neurospora crassa (strain GR-107) containing a hexahistidinyl-tagged form of Tom22 was cultured and mitochondria were isolated as described (43). Briefly, ~1.5 kg wet weight of hyphae was homogenized in 250 mM sucrose, 2 mM EDTA, 20 mM Tris pH 8.5, 1 mM phenyl methylsulfonyl fluoride (PMSF) in a Waring mixer at 4 °C.…”