Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent K m for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondriacytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.xidative phosphorylation requires transport of metabolites, including cytosolic ADP, ATP, and inorganic phosphate, across both mitochondrial membranes for F 1 F 0 -ATPase to generate ATP in the matrix. Voltage-dependent anion channel (VDAC, also called mitochondrial porin) is the most abundant protein in mitochondrial outer membrane (MOM) and is known to be primarily responsible for ATP/ADP flux across the outer membrane (1, 2). Until recently, VDAC was generally viewed as a part of the pathway for release of cytochrome c and other apoptogenic factors from the mitochondrial intermembrane space into the cytosol at the early stage of apoptosis. The recent genetic studies undermined this view (3) but still left open a lot of questions concerning the role of VDAC in MOM permeabilization in apoptosis (4-6). A conserved property of VDACs in vitro is the ability to adopt a unique fully open state and multiple states with significantly smaller conductance (7). It was demonstrated that the latter, so called ''closed states'' are impermeable to ATP but still permeable to small ions (8), including Ca 2ϩ (9). In isolated mitochondria, respiration is characterized by an apparent K m for exogenous ADP that is Ϸ10-fold lower than in permeabilize...

The mitochondrial channel, VDAC, forms large (3 nm in diameter) aqueous pores through membranes. We measured ATP flow (using the luciferin/luciferase method) through these channels after reconstitution into planar phospholipid membranes. In the open state of VDAC, as many as 2 x 10(6) ATP molecules can flow through one channel per second. The half-maximum rate occurs at approximately 75 mM ATP. The permeability of a single channel for ATP is 1.1 x 10(-14) cm3/s (about 1 cm/s after correcting for cross-sectional area), which is 100 times less than the permeability for chloride and 10 times less than that for succinate. Channel closure results in a 50% reduction in conductance, showing that monovalent ions are still quite permeable, yet ATP flux is almost totally blocked. This is consistent with an electrostatic barrier that results in inversion of the selectivity of the channel and could be an example of how large channels selectively control the flow of charged metabolites. Thus VDAC is ideally suited to controlling the flow of ATP between the cytosol and the mitochondrial spaces.

Background: Metabolites generating mitochondrial membrane potential (⌬⌿) enter through voltage-dependent anion channels (VDAC). Results: VDAC3 contributed to ⌬⌿ formation more than VDAC1/2. VDAC3 knockdown decreased ATP and NADH/NAD ϩ .