Targeting and assembly of rat mitochondrial translocase of outer membrane 22 (TOM22) into the TOM complex.
Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic alpha-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20.
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-0054, Japan.
Yasuhiko
Y
Nakamura
Hiroyuki
H
Suzuki
Masao
M
Sakaguchi
Katsuyoshi
K
Mihara
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C090857:TOM22 protein, S cerevisiae; C489971:TOMM22 protein, human