Abstract 16620: ATP Binding Cassette Protein B10 is a Putative Mitochondrial Exporter of 5-Aminolevulinic Acid (ALA) in Cardiomyoblasts
Heme is a prosthetic group required for a diversity of cellular functions including transport of diatomic gases, oxidative phosphorylation and chemical catalysis. Heme synthesis begins in the mitochondrial matrix with the production of ALA, which is then exported into the cytosol for subsequent conversions. The transporter regulating this step has not been identified yet. We hypothesized that ABCB10, an inner mitochondrial ABC protein, plays a role in ALA delivery to the cytoplasm and is essential for heme synthesis.
Results: Lentiviral knockdown of ABCB10 in cardiac myoblasts resulted in a significant reduction both in cellular and mitochondrial heme content, suggesting that ABCB10 is essential for heme synthesis. Overexpression of ABCB10 using adenovirus did not alter heme levels and activities of heme-containing proteins, indicating that ABCB10 does not mediate a rate-limiting step in the synthesis of heme. We next focused on identifying the precise step in heme synthesis that is carried out by ABCB10. First, we assessed whether ABCB10 has a role in mitochondrial iron delivery. While ABCB10 knockdown reduced total cellular iron content measured by 55Fe radiotracer, the mitochondrial 55Fe levels were not altered, suggesting that ABCB10 is not directly involved in iron transport into the mitochondria. Moreover, we observed no defect in 55Fe incorporation into protoporphyrin IX (PPIX), the precursor molecule to heme, with ABCB10 knockdown and addition of exogenous ALA, suggesting that ABCB10 is not required for the last step of heme synthesis. Finally, we examined the role of ABCB10 in the transport of ALA out of the mitochondria. To determine if ABCB10 functions as an ALA exporter, we incubated ABCB10 knockdown cells with exogenous ALA and measured heme content. While ABCB10 downregulation alone resulted in reduced heme levels, addition of ALA restored both total cellular and mitochondrial heme levels to those observed in the control group, suggesting that ABCB10 plays a role in ALA transport from mitochondria into the cytoplasm.
Conclusions: Our data suggest that ABCB10 is not required for iron delivery or incorporation into PPIX in cadiomyoblasts, but functions to export ALA out of the mitochondria for the cytosolic steps of heme biosynthetic pathway.
- © 2011 by American Heart Association, Inc.