Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched beta-structure.
TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% beta-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(DeltaN165)) as a recombinant protein with 62% beta-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched beta-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.
Suzuki
Hiroyuki
H
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
Kadowaki
Tomoko
T
Maeda
Maki
M
Sasaki
Hiroyuki
H
Nabekura
Junichi
J
Sakaguchi
Masao
M
Mihara
Katsuyoshi
K
D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C066555:Tom40 protein, S cerevisiae; C000707766:Tomm40 protein, rat