Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia

Paola Lunetti,Fabrizio Damiano,Giuseppe De Benedetto,Luisa Siculella,Antonio Pennetta,Luigina Muto,Eleonora Paradies,Carlo Marya Thomas Marobbio,Vincenza Dolce,Loredana Capobianco

doi:10.1074/jbc.m116.736876

Abstract

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia.

Highlights

Universities, and Research (MIUR) (SIR 2015, molecular and pathogenetic mechanism of congenital sideroblastic anemia associated with the SLC25A38 gene deficiency)
Functional Characterization of Recombinant Hem25p— Because it has been hypothesized that Hem25p facilitates aminolevulinic acid (ALA) production by importing glycine into mitochondria [13, 14], the recombinant and purified Hem25p was reconstituted into liposomes, and its ability to transport glycine was tested
When yeast cells are grown on glucose as the carbon source, glycine can be produced by the catabolism of threonine in the cytosolic L-threonine aldolase reaction [30]

Summary

Introduction

Universities, and Research (MIUR) (SIR 2015, molecular and pathogenetic mechanism of congenital sideroblastic anemia associated with the SLC25A38 gene deficiency). Guernsey et al [13] has characterized a subset of patients with severe non-syndromic congenital sideroblastic anemia resembling X-linked sideroblastic anemia but lacking the ALAS2 mutations [13] In this subgroup of patients the author identified several mutations in the SLC25A38 gene [13]. The recombinant SLC25A38 protein ( named GlyC, glycine carrier) overexpressed in bacteria and reconstituted into phospholipid vesicles transported glycine, and its gene was able to rescue the growth defect in hem25⌬ cells. This is the first time that the protein responsible for the uptake of glycine into mitochondria has been identified at the molecular level

Methods

Results

Conclusion