Blood, 15 December 2002, Vol. 100, No. 13, pp. 4678-4678
CORRESPONDENCE
To the editor:
Heme inhibits the mitochondrial import of coproporphyrinogen
oxidase
Heme has a variety of catabolic and regulatory functions within
cells. It serves as a prosthetic group in hemeproteins, such as
cytochromes, globins and catalases. The need for heme is greatest during erythropoiesis, where large amounts of heme are required in the
synthesis of hemoglobin. The induction of heme biosynthesis is an early
event in erythroid differentiation, and the expression of each enzyme
of the pathway increases sequentially. Among the 8 enzymes of the
pathway, 
-aminolevulinate synthase (ALAS) is the rate-limiting
enzyme. Therefore, the finely tuned regulation of the synthesis of
ALAS, or an erythroid-specific form (ALSE), is necessary for erythroid
differentiation. Several mechanisms involved in this regulation
have been reported.1,2 Among them, feedback inhibition of
transcription of the gene and mitochondrial import of the protein by
heme seem to be important.2 Recently, coproporphyrinogen
oxidase (CPO), the sixth enzyme of the pathway, has been reported to
represent rate-limiting steps downstream from ALASE in the regulation
of heme biosynthesis.3,4 The regulation of the synthesis
of CPO has been reported,5 but the regulation by heme has
not been reported. Here, we examined whether heme inhibits the
mitochondrial import of CPO as in the case of ALASE.
Full-length human CPO cDNA was obtained by polymerase chain
reaction (PCR) using the human liver cDNA library as template. The
forward and reverse primers used were 5'-TGCGGGAACATGGCCTTGCAGCTGGG-3' and 5'-CTCCAAACCCCTGCACAGCCATTCTG-3', respectively. PCR products were
then cloned in vitro in the transcription vector pGEM-5Z under the
control of the SP6 RNA polymerase. Using this cDNA, the precursor protein of CPO (preCPO) was synthesized in the
transcription-translation-coupled system in the presence of
35S-methionine and then was imported into rat-liver
mitochondria as previously reported.6 We used
Sub9-DHFR in which the presequence of subunit 9 (Sub9) of the
F0-ATPase (residue 1-69) was fused at the C-terminus with
dihydrofolate reductase (DHFR) and a precurser protein of AAC (ATP/ADP
carrier; preAAC) as controls. The mitochondrial imports of these
precursor proteins are known not to be influenced by heme.
Transport of preCPO into isolated rat mitochondria was 99%
inhibited in the presence of 20 µM exogenous hemin (Figure
1). In contrast, the transport of Sub9-DHFR and preAAC, assayed under identical conditions, was not inhibited at low hemin concentrations and
was below 20% inhibited by 20 µM hemin (Figure 1). These results clearly indicate that heme inhibits the mitochondrial import of preCPO.
Therefore, it is likely that heme regulates its own synthesis by
inhibiting the import of not only ALASE but also CPO. The mechanisms of
this feedback inhibition of mitochondrial import of CPO by heme are not
clear. The heme,2 regulatory motif, which was identified in
the presequence of ALASE and was shown to be involved in the feedback
inhibition by heme,2 was not identified in preCPO.
Therefore, heme seems to regulate the mitochondrial import of CPO by
the mechanisms that differ from that involved in ALASE. These results
shed light on the field of clarifying the importance of heme on the
regulation of the synthesis of CPO.
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| Figure 1.
Inhibition of the mitochondrial import of preCPO by high
hemin concentration.
Import reaction was performed in the presence or absence of hemin at
the indicated concentrations for 30 minutes at 30°C. Then
mitochondria were treated with trypsin and CPO, and Sub9-DHFR and
AAC were analyzed by fluorography. The bands of mature CPO,
mature Sub9-DHFR, and AAC were quantified by densitometry of exposed
film.  represents CPO;  , Sub9-DHFR; and  , AAC.
|
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Shinji Susa, Makoto Daimon, Hideyu Ono, Song Li, Tadashi Yoshida, and Takeo Kato
Correspondence: Makoto Daimon, The Third
Department of Internal Medicine, Yamagata University School of
Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan; e-mail:
mdaimon{at}med.id.yamagata-u.ac.jp
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