[in Japanese]

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In vertebrates, the rate-limiting step of heme biosynthesis is catalyzed by two isoforms of 5’- aminolevulinate synthase (ALAS) in a tissue-specific manner. The nonspecific isoform ALAS1 is expressed in all cells and undergoes heme-dependent degradation by mitochondrial matrix proteases, whereas ALAS2 is the erythroid-specific isoform that is strictly and stably expressed in erythroid cells to supply excess heme for hemoglobin production. Unlike that of ALAS1, the regulation of ALAS2 protein turnover in mitochondria has yet to be unequivocally elucidated. In this study, we found that FLAG-tagged ALAS2 (ALAS2F) expressed in a nonerythroid human cell line has a longer half-life than FLAG-tagged ALAS1, although it similarly forms complexes with mitochondrial matrix proteases under conditions of excess heme. To identify the possible proteins stabilizing ALAS2, we analyzed ALAS2F protein immunoprecipitates using mass spectrometry and identified several mitochondrial chaperone proteins, including HSPA9. Knockdown or chemical inhibition of HSPA9 caused a decrease in the ALAS2F protein level, which was assumed to result from increased degradation of ALAS2F, implying that HSPA9 plays a vital role in stabilizing the ALAS2 protein to resist degradation in the mitochondrial matrix.

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Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the rate-limiting enzyme of the heme biosynthetic pathway in erythroblasts. ALAS2 is essential for supplying heme to produce hemoglobin in developing erythroblasts. While it has been reported that the gene expression of ALAS2 is regulated at the transcriptional and the translational steps, the post-translational regulation of ALAS2 protein is not well understood. In this study, we examined the protein complex of FLAG-tagged ALAS2 (ALAS2F) using immunoprecipitation followed by mass spectrometry and identified several mitochondrial proteins in the complex of ALAS2F. We also confirmed the presence of these proteins in the complex of ALAS2F by Western blot analysis, and we speculated on the novel role of one of these identified proteins on the post-translational regulation of ALAS2 protein. Further studies on the role of these proteins in the regulation of ALAS2 protein will reveal the precise mechanisms of the post-translational regulation of ALAS2 in erythroid cells.

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