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, δ-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 of³⁵S-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 (Figure1). 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.