CHARACTERISATION OF MITOCHONDRIAL TARGETING OF WILD TYPE AND MUTANT (L88Q) HUMAN COPROPORPHYRINOGEN OXIDASE

Van Der Merwe D, Roberts AG, Badminton MN

Departments of Medical Biochemistry and Immunology, University Hospital of Wales and School of Medicine, Cardiff University, Cardiff

Coproporphyrinogen Oxidase (CPO), the sixth enzyme in the haem biosynthetic pathway, is located in the mitochondrial intermembrane space.  Partial deficiency of CPO underlies hereditary coproporphyria, an autosomal dominant acute porphyria. The 453 amino acid apo-CPO is imported into the mitochondrial intermembrane (IM) space through a cleavable 110 amino acid leader peptide, although this has not been confirmed in living cells. Analyses of hydrophobicity and secondary structure conformational parameters in both human and mouse CPO suggests that the leader sequence is a bipartite structure, consisting, in human CPO, of a positively charged matrix-targeting signal region (residues 1-69) followed by an extended hydrophobic sorting region (residues 70-103) that may direct the protein to the inter-membrane space.  We have fused human CPOs, containing N-terminal and C-terminal deletions, to the amino terminus of yellow fluorescent protein (YFP) and have imaged mitochondrial targeting by fluorescence and confocal microscopy.  Here we show that amino acid residues 1-69 contain all the information necessary to target YFP to the mitochondria in living cells. Residues 70-110 were not, by themselves, able target YFP. A pathogenic mutation, L88Q, identified in a patient with HCP does not prevent targeting. We are currently investigating the role of the hydrophobic region in localising CPO to the inter-membrane space.