title: Trypanosome alternative oxidase possesses both an N-terminal and internal mitochondrial targeting signal.
abstract: Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.
authors.affiliation: Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee, USA.
authors.forename: Vanae
authors.initials: V
authors.lastname: Hamilton
authors.forename: Ujjal K
authors.initials: UK
authors.lastname: Singha
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Smith
authors.forename: Ebony
authors.initials: E
authors.lastname: Weems
authors.forename: Minu
authors.initials: M
authors.lastname: Chaudhuri
mesh_terms: D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D015870:Gene Expression; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D010088:Oxidoreductases; D013762:Tetrahydrofolate Dehydrogenase