Post-translational control of erythroid-specific ALA synthase: Domains involved in mttochondrial targeting, translocation and prevention from aggregation

Kazumichi Furuyama, Hideo Harigae, Shigeru Sassa

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Erythroid specific 5-aminolevulinate synthase (ALAS-E) supplies heme for hemoglobin synthesis in erythroid cells. While several mutants of ALAS-E gene have been reported to be responsible for X-linked sideroblastic anemia, there is no information on ALAS-E mutation in other forms of hereditary sideroblastic anemias. We have previously demonstrated that ALAS-E associates with the β-subunit of succinyl CoA synthetase both in cytoplasm and mitochondria, and a certain mutant of ALAS-E which fails in this association results in a marked deficiency in mitochondria! ALAS-E (K. Furuyama & S. Sassa, J. Clin. Invest. 105:757, 2000). In order to define the domain involved in mitochondrial targeting and translocation, we prepared several deletion mutants of the presequence of ALAS-E and expressed them in QT6 quail fibroblasts. These constructs had either 10, 20, or 40 amino acids (aa) deletion at the N-terminus of ALAS-E protein, which we termed pAEdelNl, pAEdelN2, or pAEdelN4, respectively. When the wild type ALAS-E was expressed in QT6 cells, intense red fluorescence was observed under UV irradiation due to a large amount of porphyrin accumulation in the cell. When pAEdelNl was expressed, only a small amount of porphyrins was observed, and no porphyrins were observed when pAEdelN2, or pAEdelN4 was expressed, suggesting that the first 10 aa are critical in mitochondrial translocation of ALAS-E. Next, we made expression vectors using pAEdelNl, pAEdelN2, or pAEdelN4, fused with GFP, which we termed pAEdelNl-OFP, pAEdelN2-GFP, or pAEdelN4-GFP, respectively. When GFP fused with the wild type presequence of ALAS-E was expressed in QT6 cells, distribution pattern of green fluorescence was granular and coincided with the localization of mitochondria as observed by fluorescence microscopy. In contrast, when pAEdelN2GFP was expressed, fluorescence distribution was aggregated, and aggregates were independently localized from mitochondria. pAEdelN4-GFP transfectants showed yet an entirely different fluorescence distribution pattern which was diffuse in the cytoplasm. These data suggest that the first 10 through 20 aa of the presequence of ALAS-E contain an important domain involved in the mitochondria! targeting. In addition, protein interaction at the first 10 aa of the presequence appears to be essential in preventing the ALAS-E precursor protein from aggregation in the cytoplasm.

Original languageEnglish
Pages (from-to)4a-4b
Issue number11 PART I
Publication statusPublished - 2000

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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