1999 Fiscal Year Final Research Report Summary
Molecular Mechanism of Posttranscriptional modification by Heme
| Project/Area Number |
10680595
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | KINKI UNIVERSITY |
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Principal Investigator |
MUNAKATA Hiroshi Kinki University, School of Medicine, Professor, 医学部, 教授 (90111294)
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| Co-Investigator(Kenkyū-buntansha) |
YOSIDA Koji Kinki University, School of Medicine, Research Associate, 医学部, 助手 (60230736)
YAMAMOTO Kazuhiko Kinki University, School of Medicine, Research Associate, 医学部, 助手 (00166787)
SAITO Akio Kinki University, School of Medicine, Assistant Professor, 医学部, 講師 (40153788)
AMEMIYA Kana Kinki University, School of Medicine, Research Associate, 医学部, 助手 (30195929)
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| Project Period (FY) |
1998 – 1999
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| Keywords | 5-aminolevulilate synthase / mitochondria / intracellular protein sorting / heme-regulatory motif |
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| Research Abstract |
5-aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS-N) is regulated by heme. Recently, a short amino acid sequence, the heme regulatory motif (HRM), has been shown to be involved in the hemin inhibition of protein transport in vitro. To elucidate the role of HRM in the heme regulation of ALAS transport in vivo, we constructed a series of mutants of rat ALAS-N in which the specific cysteine residues within the HRMs were converted to serines by site-directed mutagenesis. Wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, followed by analyses of the mitochondrial import of the enzymes. The heme inhibition, which was observed in the wild-type ALAS-N, abolished completely when all the three HRMs in the enzyme were mutated, indicating that the HRM is actually required for the heme inhibition of ALAS-N transport within the cells. In contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS (ALAS-E) under the comparable experimental conditions. Mitochondrial import of the deletion mutant of ALAS-N in which 40 amino acids between HRM2 and HRM3 are removed was not affected by heme. Then, fusion protein of presequnce of ALAS-E and mature enzyme of ALAS-N was made. Heme inhibited the mitochondrial import of the fusion enzyme. These results may reflect the difference in the physiological function between two ALAS isoforms.
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Research Products
(6 results)
