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 Cterminal five-residue segment (C-segment) contained the information required for mitochondrialtargeting. 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.

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.

Mitochondrial outer membrane proteins are synthesized without a cleavable presequence but instead contain segments responsible for mitochondrial targeting and membrane integration within the molecule: the transmembrane segment (TMS) and N-or C-terminal flanking segment. We analyzed targeting and integration of Tom5, a C-tail anchor protein associated with the preprotein translocase of the outer membrane, to the yeast mitochondrial outer membrane in vivo using green fluorescent protein as the reporter and compared the signal with other signals for proteins dispersed in the membrane. The functional assembly of Tom5 into the TOM complex was assessed by blue native PAGE and complementation of temperature-sensitive ⌬tom5 cells. Correct targeting and assembly required (i) an appropriate length TMS rather than hydrophobicity, (ii) a proline residue located at correct position in the TMS and specific residues near the proline, and (iii) that, in contrast to proteins dispersed in the outer membrane, the positive C-terminal segment was dispensable. Based on these findings, we constructed green fluorescent protein fusions with a C-terminal TMS in which the deduced sequences (minimum: Ser-Pro-Met) were inserted at an appropriate position within artificial Leu-Ala repeats. They were targeted to mitochondria and complemented the temperature-sensitive growth phenotype of ⌬tom5 yeast cells. The membrane-targeting mechanism of Tom5 appears to be distinct from that for proteins that are dispersed in the outer membrane.Most mitochondrial proteins are synthesized in the cytosol and post-translationally transported to mitochondrial subcompartments. In contrast to proteins destined for the matrix or inner membrane (1-3), the targeting mechanism and the topogenesis of mitochondrial outer membrane proteins remains poorly understood, although targeting signals have been analyzed for several outer membrane proteins (4 -11).Mitochondrial outer membrane proteins are synthesized without a cleavable presequence, and the mitochondrial-targeting and topogenic information is contained within the mature protein sequence; usually within ␣-helical transmembrane segments (TMS) 1 and flanking regions (12, 13). The import receptors of the preprotein translocase of the mitochondrial outer membrane (TOM (translocase of outer membrane) complex) (3, 14), Tom70 (15, 16), and Tom20 (8, 17) are anchored to the membrane through the N-terminal TMS in the Nin-Cout orientation. Tom22, which functions both as the preprotein receptor and organizer of the TOM complex, is anchored to the membrane in the Nout-Cin orientation through the internal TMS (7, 18 -20). Tom5, Tom6, and Tom7 are anchored to the outer membrane through the C-terminal TMS (tail-anchored protein). Tom5 functions as the connecting link between import receptors and the translocation channel Tom40 (21). Tom6 (22-24) and Tom7 (25) function as modulators of the TOM channel (26). Tom40, like porin, is predicted to be a ␤-barrel protein spanning the outer membrane by 12-14 antiparallel ␤-strands (27-30...