title: Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis.
abstract: In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.
authors: From the Department of Molecular Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 and.
authors: Yoshiko
authors: Y
authors: Kubota
authors: From the Department of Molecular Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 and.
authors: Kazumi
authors: K
authors: Nomura
authors: the Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors: Yasutake
authors: Y
authors: Katoh
authors: From the Department of Molecular Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 and.
authors: Rina
authors: R
authors: Yamashita
authors: From the Department of Molecular Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 and.
authors: Kiriko
authors: K
authors: Kaneko
authors: From the Department of Molecular Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 and furuyama@iwate-med.ac.jp.
authors: Kazumichi
authors: K
authors: Furuyama
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000235:genetics / Q000378:metabolism*; D049069:ATP-Dependent Proteases / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D059748:Proteolysis
chemical_list: D024101:Mitochondrial Proteins; D006418:Heme; D000624:5-Aminolevulinate Synthetase; D049069:ATP-Dependent Proteases; C000595973:LONP1 protein, human; C568646:ClpP protein, human; D049071:Endopeptidase Clp; C528384:CLPX protein, human
keywords: 5-aminolevulinate synthase; ATP-dependent protease; heme; mass spectrometry (MS); mitochondria; post-translational modification (PTM); protein degradation