Targeting and Assembly of Mitochondrial Tail-anchored Protein Tom5 to the TOM Complex Depend on a Signal Distinct from That of Tail-anchored Proteins Dispersed in the Membrane*

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 Δtom5 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 Δtom5 yeast cells. The membrane-targeting mechanism of Tom5 appears to be distinct from that for proteins that are dispersed in the outer membrane.