The DNA helicase, Hmi1p, is transported into mitochondria by a C-terminal cleavable targeting signal

We have identified a novel mitochondrial targeting signal in the precursor of the DNA helicase Hmi1p of Saccharomyces cerevisiae that is located at th e C terminus of the protein. Similar to classical N-terminal presequences, this C-terminal targeting signal consists of a stretch of positively charge d amino acids that has the potential to form an amphipathic ct-helix. Delet ion of the C-terminal 36 amino acids of helicase resulted in loss of import into mitochondria, while deletion of the N-terminal 40 amino acids had no effect. When C-terminal regions of the helicase were placed at the C termin us of a passenger protein, dihydrofolate reductase, the resulting fusion pr oteins were directed into the mitochondrial matrix, and the C-terminal regi on of helicase became proteolytically processed. Import of helicase occurs in a C- to N-terminal direction; it requires a membrane potential and the T IM17-23 translocase together with mitochondrial Hsp70. Helicase is the only mitochondrial matrix protein identified thus far with a cleavable targetin g signal at its C terminus.