CHARGED AMINO-ACIDS AT THE CARBOXYL-TERMINAL PORTIONS DETERMINE THE INTRACELLULAR LOCATIONS OF 2 ISOFORMS OF CYTOCHROME B(5)

Outer mitochondrial membrane cytochrome b(5) (OMb), which is an isofor m of cytochrome b(5) (cyt b(5)) in the endoplasmic reticulum, is a typ ical tail-anchored protein of the outer mitochondrial membrane. We clo ned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitoc hondrial targeting signal at the amino terminus. To identify the regio n responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellul ar localization of the expressed proteins was analyzed. The deletion o f more than II amino acid residues from the carboxyl-terminal end of O Mb abolished the targeting of the protein to the mitochondria. When th e carboxyl-terminal 10 amino acids of OMb were fused to the cyt b(5) t hat was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the ca rboxyl-terminal 10 amino acid residues of OMb have sufficient informat ion to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 a mino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b(5), in which the acidi c amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem tha t charged amino acids in the carboxyl-terminal portion of these protei ns determine their locations in the cell.