A 24-residue presequence localizes human factor B to mitochondria.
We reported previously that the human factor B precursor is a 215-amino acid polypeptide, the first 40 amino acid residues of which function as a mitochondrial targeting presequence [G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097-6103]. Confocal microscopy of live HEK293 cells, transiently transfected with factor B constructs tagged at the C-terminus with green fluorescent protein (GFP) revealed that either a 40- or 25-residue presequence localized factor B to mitochondria. Indirect immunofluorescent labeling of fixed, permeabilized HEK293 cells that were transiently transfected with a construct lacking a presequence, showed diffuse, intracellular staining that was consistent with targeting of ectopically expressed factor B to cellular compartments distinct from the mitochondria. Mutants in which either Met(-25) or both Met(-25)/Met(-24) residues of the presequence were deleted exhibited decreased or undetectable levels, respectively, of the GFP-tagged factor B. The factor B presequence alone was shown to target a reporter polypeptide GFP to mitochondria. Our studies, therefore, demonstrate that a 24-residue presequence is sufficient to localize factor B to mitochondria, and suggest that the human factor B precursor is a 199-amino acid polypeptide.
Belogrudov
Grigory I
GI
Department of Physiology, David Geffen School of Medicine, University of California at Los Angeles, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA. gbelo@ucla.edu
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D011415:Complement Factor B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D051381:Rats
D033681:Mitochondrial Membrane Transport Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011415:Complement Factor B; D025261:Mitochondrial Proton-Translocating ATPases