Abstract
Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.
Highlights
Introduction of the vector carrying ratTom20 cDNA into the mutant cells complemented the growth defect of the cells on a nonfermentable carbon source the growth rate was slightly slower than that of wild-type cells (Figs. 4A-C)
Isolation of Rat Tom20 cDNA—A search of the EBI Data Bank revealed that a human cDNA with an open reading frame encoding a protein of 145 amino acid residues (DDBJ, accession number D13641) exhibited a significant homology to Tom20 of Saccharomyces cerevisiae and Neurospora crassa
The membrane-anchor mutant Tom20⌬2–18 was expressed at about 30% of the level of wildtype Tom20 but was unable to complement the growth defect of ⌬tom20 yeast cells (Table I). These results suggest that the linker domain and the membrane anchorsegment are essential for the function of rat Tom20, whereas the tetratricopeptide repeat (TPR) motif as well as the C-terminal acidic amino acid cluster are not
Summary
Materials—MSF and hsp were purified from rat liver cytosol according to Hachiya et al (3) and Deshaies et al (15), respectively. Mitochondria (50 g) isolated from ⌬tom yeast cells expressing rat Tom20N69 were incubated with or without pre-immune IgGs (100 g) or IgGs against yeast Tom (ammonium sulfate-fractionated, 100 g) or rat Tom (affinity purified, 4 g) at 0 °C for 30 min in 50 l of the import buffer consisting of 10 mM HEPES-KOH, pH 7.5, 250 mM sucrose, 1 mM ATP, 1 mM reduced glutathione, 5 mM magnesium acetate, 20 mM sodium succinate, and 60 mM potassium acetate, washed once, and subjected to the import at 30 °C for 60 min using 125I-pAd1⁄7MSF complex or 125I-pAd1⁄7hsp complex. Rat liver mitochondria (100 g) were treated with 100 g of pre-immune IgGs or 50 g of anti-rat Tom IgGs at 0 °C for 60 min in 50 l of the import buffer, washed once with the import buffer, and subjected to the import using pAd-hsp or pAd-MSF-hsp as the substrate in which all of the components were labeled with 125I (20). Yeast lysates were prepared according to Yamazaki et al (23)
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