Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria.
The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106-125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitro import of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.
Yano
M
M
Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan. masa@gpo.kumamoto-u.ac.jp
Kanazawa
M
M
Terada
K
K
Takeya
M
M
Hoogenraad
N
N
Mori
M
M
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D019556:COS Cells; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C084008:TOMM20 protein, human; C489998:Tomm20 protein, rat; D010450:Endopeptidases