Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

J Biol Chem. 2016 Sep 23;291(39):20516-29. doi: 10.1074/jbc.M116.719161. Epub 2016 Aug 5.


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.

Keywords: 5-aminolevulinate synthase; ATP-dependent protease; heme; mass spectrometry (MS); mitochondria; post-translational modification (PTM); protein degradation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Aminolevulinate Synthetase / genetics
  • 5-Aminolevulinate Synthetase / metabolism*
  • ATP-Dependent Proteases / genetics
  • ATP-Dependent Proteases / metabolism
  • Amino Acid Motifs
  • Binding Sites
  • Endopeptidase Clp / genetics
  • Endopeptidase Clp / metabolism
  • Heme / genetics
  • Heme / metabolism*
  • Hep G2 Cells
  • Humans
  • Mitochondria / enzymology*
  • Mitochondria / genetics
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Oxidation-Reduction
  • Proteolysis*


  • Mitochondrial Proteins
  • Heme
  • 5-Aminolevulinate Synthetase
  • ATP-Dependent Proteases
  • LONP1 protein, human
  • ClpP protein, human
  • Endopeptidase Clp
  • CLPX protein, human