Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase.
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 (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.
Munakata
Hiroshi
H
Department of Biochemistry, School of Medicine, and Life Science Institute, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama, 589-8511. munakata@med.kindai.ac.jp
Sun
Ji-Ying
JY
Yoshida
Koji
K
Nakatani
Tatsuya
T
Honda
Eiko
E
Hayakawa
Sumio
S
Furuyama
Kazumichi
K
Hayashi
Norio
N
D000624:5-Aminolevulinate Synthetase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011136:Polysorbates / Q000494:pharmacology; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011784:Quail; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014162:Transfection
D003902:Detergents; D011136:Polysorbates; D020033:Protein Isoforms; D012967:Sodium Dodecyl Sulfate; D006418:Heme; D006427:Hemin; D000624:5-Aminolevulinate Synthetase