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Vol. 13, Issue 5, 1615-1625, May 2002
Department of Molecular Biology, Graduate School of Medical
Sciences, Kyushu University, Fukuoka 812-8582, Japan
We analyzed the signal that directs the outer membrane protein with
the C-terminal transmembrane segment (TMS) to mammalian mitochondria by
using yeast Tom5 as a model and green fluorescent protein as a
reporter. Deletions or mutations were systematically introduced into
the TMS or the flanking regions and their intracellular localization in
COS-7 cells was examined using confocal microscopy and cell
fractionation. 1) Three basic amino acid residues within the C-terminal
five-residue segment (C-segment) contained the information required for
mitochondrial-targeting. Reduction of the net positive charge in this
segment decreased mitochondrial specificity, and the mutants were
distributed throughout the intracellular membranes. 2) Elongation of
the TMS interfered with the function of the C-segment and the mutants
were delivered to the intracellular membranes. 3) Separation of the TMS
and C-segment by linker insertion severely impaired mitochondrial
targeting function, leading to mislocalization to the cytoplasm. 4)
Mutations or small deletions in the region of the TMS flanking the
C-segment also impaired the mitochondrial targeting. Therefore, the
moderate length of the TMS, the positive charges in the C-segment, and
the distance between or context of the TMS and C-segment are critical
for the targeting signal. The structural characteristics of the
signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.
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