FUNCTIONAL-ANALYSIS OF HUMAN MITOCHONDRIAL RECEPTOR TOM20 FOR PROTEINIMPORT INTO MITOCHONDRIA

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membran e-anchor segment, a linker segment rich in charged amino acids, a tetr atricopeptide repeat motif a glutamine-rich segment, and a c-terminal segment, To assess the role of each segment, four C-terminally truncat ed mutants of the human receptor (hTom20) were constructed, and the ef fect of their overexpression in COS-7 cells was analyzed, Expression o f a mutant lacking the tetratricopeptide repeat motif inhibited preorn ithine transcarbamylase (pOTC) import to the same extent as the wild-t ype receptor. Thus, overexpression of the membrane-anchor and the link er segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end o f 20 amino acid residues stimulated import of pOTC-green fluorescent p rotein (GFP), a fusion protein in which the presequene of pOTC was fus ed to green fluorescent protein. On the other hand, expression of muta nts lacking either the glutamine-rich segment or larger deletions inhi bited pOTC-GFP import. In. vitro import of pOTC was inhibited by the w ild-type hTom20 and the mutant lacking the C-terminal end, but much le ss strongly by the mutant lacking the glutamine-rich segment. On the o ther hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderate ly decreased by the deletion of the glutamine-rich segment, whereas pO TC-GFP binding was completely lost by this deletion. Binding of pOTCN- GFP a construct that contains am 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 t hat this region is important for mitochondrial aggregation. The differ ent behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants i s explicable on the basis of the conformation of the precursor protein s.