The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70–Tim44 driving system. By blue native electrophoresis, we identify an ∼90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence‐containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

IntroductionThe hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes regulating mammalian oxygen homeostasis. 1 Among others, the corresponding gene products are involved in erythropoiesis, iron metabolism, angiogenesis, control of blood flow, glucose uptake and glycolysis, pH regulation, and cell-cycle control. 2 HIF-1 is a ␣ 1 ␤ 1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements. 3 Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein (pVHL) targets the HIF-1␣ subunit for rapid ubiquitination and proteasomal degradation. 4 Binding of the pVHL tumor suppressor protein requires the modification of HIF-1␣ by prolyl-4-hydroxylation at prolines 402 and 564 of human HIF-1␣. [5][6][7][8] A family of 3 oxygen-and iron-dependent prolyl-4-hydroxylases called PHD1, PHD2, PHD3, or HPH3, HPH2, HPH1, respectively, has been shown to hydroxylate HIF␣. 9,10 A fourth member, called PH-4, regulates HIF-1␣ in overexpression conditions only. 11 Thus, limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 This unusual mechanism of protein regulation provides the basis for the very rapid HIF-1␣ response to hypoxia. 13 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF-1␣ by factor inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity following a further decrease in oxygen availability. 14,15 Among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which coordinately regulate iron metabolism. [16][17][18][19][20] Increased iron uptake, release from the liver, plasma transport, and uptake in the bone marrow are essential to sustain the erythropoietic function of erythropoietin, the prototype HIF-1 target. Ceruloplasmin is a multicopper plasma protein containing ferroxidase activity necessary for Fe 3ϩ saturation of transferrin. 21 Hereditary aceruloplasminemia in humans as well as targeted deletion of the ceruloplasmin gene (Cp) in mice results in iron metabolism disorders characterized by anemia, hepatic iron overload, and neurodegeneration, demonstrating a tight connection between copper and iron metabolism. [22][23][24][25][26] Iron deficiency has been known for more than a decade to induce erythropoietin gene expression and HIF-1␣ protein stabilization. 27 Nowadays, these results are most likely explained by inactivation of the iron-dependent protein hydroxylases PHD1 to 3 and FIH. 12 Iron deficiency also results in mRNA induction of ceruloplasmin by HIF-1-dependent promoter activation and subsequent transcriptional up-regulation of the Cp gene. 20 Materials and methods Cell lines and cell cultureAll cell lines were cultured in Dulbecco modified Eagle medium (high glucose) as described previously. 29 Oxygen partial pressures in the hypoxic workstation (InVivO 2 -400; Ruskinn Technology, Leeds, United Kingdom) or in the incubator (M...