Biochem. J. (2005) 386
(381–386) (Printed in Great Britain)
Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase
Tamara A. DAILEY, John H. WOODRUFF and Harry A. DAILEY1
Departments of Microbiology, Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, University of Georgia, Athens, GA 30602-7229, U.S.A.
The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haem-binding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haem-regulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.
Key words: aminolaevulinate, ferrochelatase, haem, mitochondria, protoporphyrinogen, targeting.
Abbreviations used: ALA, aminolaevulinic acid; ALAS, 5-aminolaevulinate synthase; CPO, coproporphyrinogen oxidase; FC, ferrochelatase; GFP, green fluorescent protein; PPO, protoporphyrinogen oxidase; for brevity, the single-letter system for amino acids has been used; C8, for example means Cys8.
1To whom correspondence should be addressed (email hdailey@arches.uga.edu).
The nucleotide sequence data reported for the mouse CPO exon 1 sequence will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession no. AB011243. The resulting product, when sequenced, identified 12 base miscalls and a frameshift. The corrected sequence has been submitted to GenBank® accession no. AY382578.
Received 6 April 2004/11 October 2004; accepted 14 October 2004
Published as BJ Immediate Publication 14 October 2004, DOI 10.1042/BJ20040570
The Biochemical Society, London ©2005
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