Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins

Abstract

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.

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“…Thus, the Tim23 channel is not simply a cylindrical pore; rather, its internal and external diameters differ considerably. At its narrowest point, the Tim23 channel is smaller than both the Tom40 channel 5,6,26 and the functional translocon of the endoplasmic reticulum (∼20-50 Å) [30][31][32] but is similar to the average diameter of the polypeptide exit channel of the ribosome large subunit (15 Å) 33 . The restriction zone diameter of the Tim23 channel is just large enough to accommodate one polypeptide chain in an α-helical conformation but not large enough to contain two α-helices at the same time.…”

“…We denatured these with urea and purified Tim23 to homogeneity (Fig. 1a, lane 5), then renatured the protein by diluting the urea in the presence of the nonionic detergent nonanoyl-N-methylglucamide (Mega-9) and azolectin and reconstituted it into liposomes 5 . We fused the liposomes with a planar bilayer and detected single-channel currents (Fig.…”

“…Thus, like Tom40 (ref. 5), recombinant Tim23 may achieve at least partial translocation of the preprotein.…”

“…The diameter of the Tom40 translocation pore has been experimentally estimated at ∼20-22 Å, indicating that a polypeptide in an α-helical conformation can easily be translocated through it and that two α-helices (for example, a preprotein in a loop formation) could even be translocated together 5,6,26 . The rela- In the presence of preimmune antibodies, the current-voltage relationship was superimposable with the control trace.…”

“…Two distinct protein complexes are essential for this process: the translocase of the outer membrane (TOM complex) and a translocase of the inner membrane specific for preproteins containing a mitochondrial presequence (TIM23 complex) [1][2][3][4] . The essential protein Tom40 forms a continuous hydrophilic channel in the outer membrane that allows preproteins to pass across the membrane 5,6 . Other known Tom components function as receptors for preprotein recognition or in the assembly and stability of the TOM complex.…”