title: N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells.
abstract: Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria.
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
authors.forename: Dagmara I
authors.initials: DI
authors.lastname: Kisiela
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Aulik
authors.forename: Dhammika N
authors.initials: DN
authors.lastname: Atapattu
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Czuprynski
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D019295:Computational Biology; D047468:Immunoprecipitation; D016978:Mannheimia haemolytica / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D001427:Bacterial Toxins; D024101:Mitochondrial Proteins