Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane.
We cloned a approximately 70 kDa rat mitochondrial outer membrane protein (OM70) with a sequence identity of 28.1% and 20.1% with N. crassa and S. cerevisiae Tom70, respectively. Even with this low sequence identity, however, the proteins share a remarkable structural similarity: they have 7-10 tetratricopeptide repeat motifs and are anchored to the outer membrane through the N-terminal transmembrane domain with the bulk portion located in the cytosol. Antibodies against OM70 inhibited import of preproteins, such as the ADP/ATP carrier and rTOM40, that use internal targeting signals but not the import of cleavable presequence-containing preproteins. Blue native gel electrophoresis and immunoprecipitation of digitoninsolubilized mitochondrial outer membranes revealed that OM70 was loosely associated with the approximately 400 kDa translocase complex of the mitochondrial outer membrane, which contains rTOM22 and rTOM40. A yeast two-hybrid system demonstrated that OM70 interacted with rTOM20 and rTOM22 through the cytoplasmic domains. Thus, OM70 is a functional homologue of fungal Tom70 and functions as a receptor of the preprotein import machinery of the rat mitochondrial outer membrane. Furthermore, the N-terminal 66 residue region of OM70, which comprises a hydrophilic 41 residue N-terminal domain, a 22 residue transmembrane domain and three arginine residues, is sufficient to act as a mitochondria-targeting signal, and the arginine cluster is crucial for this function.
Suzuki
Hiroyuki
H
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
Maeda
Maki
M
Mihara
Katsuyoshi
K
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins; D002451:Cell Compartmentation / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000166:cytology / Q000201:enzymology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure
D001426:Bacterial Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D000069816:SEC Translocation Channels; C467795:Tomm70 protein, rat; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins