Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia

Abstract

The first and the rate-limiting enzyme of heme biosynthesis is δ-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the β subunit of human ATP-specific succinyl CoA synthetase (SCS-βA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-βA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-βA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-βA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria. J. Clin. Invest. 105:757-764 (2000).Using the yeast 2-hybrid system, we examined possible association of human ALAS-E with a factor that may influence the level and activity of ALAS-E in mitochondria. Our findings demonstrated that there is specific association of human ALAS-E with the human β subunit of A-SCS (hSCS-βA). In addition, our findings suggest that this association may be an important process in normal mitochondria, and its failure may be responsible for some cases of pyridoxine-refractory XLSA. Yeast 2-hybrid assay. All vectors and yeast strains that were used for yeast 2-hybrid assays were available in the MATCHMAKER Two-Hybrid System 2 (CLONTECH Laboratories Inc.). This is a complete GAL4-based 2-hybrid system that provides a transcriptional assay for detecting specific protein-protein interactions in yeast. The pAS2-1 vector is used for expressing protein fused with the GAL4 DNA-binding domain, whereas pACT2 vector is used for expressing protein fused with the GAL4 DNA-activation domain. Because both pAS2-1 and pACT2 have a nutritional marker for selection (tryptophan for pAS2-1 and leucine for pACT2), we used a minimal synthetic dropout medium (SD medium) that lacked tryptophan and leucine for selection of transformants. Because the association of each protein activates the HIS3 reporter gene that has been integrated into the genome of the Y190 strain, all yeast 2-hybrid assays were performed using SD agar plates devoid of tryptophan, leucine and histidine, but supplemented with 40 mM of 3-amino-1,2,4-triazole (3AT) (SD/-Trp/-Leu/-His/+3AT). MethodsTo construct an expression plasmid that encodes a bait protein as a fusion protein with GAL4 DNA-binding domain, human ALAS-E cDNA was digested with AflII, its sticky end was filled using Klenow DNA polymerase, and then it was digested with BamHI. Fragments were then isolated and subcloned into pAS2-1 expression vector. This was digested with NdeI, its sticky end was filled...

X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

“…Expression and purification of the MBP-SUCLA2 fusion protein was similar to that of ALAS2 above and used previously established optimal buffer conditions (22) but omitted the cleavage and final Superose 12 gel filtration steps. Four tubes, each containing 5 ml of LB medium with 100 g/ml ampicillin, were started from a glycerol stock of the SUCLA2-transformed cells with shaking at 37°C overnight.…”

“…Assuming the band staining density was proportional to protein concentration, the mol of ALAS2 bound/mol of SUCLA2 was 1.04, whereas the minor bands ranged from 0.05 to 0.4 (the band at 42 kDa was maltose-binding protein, which does not bind to ALAS2 or SUCLA2). Thus, it is highly unlikely that any of the minor bands was responsible for the observed ALAS2 binding, especially given that it has been previously shown by antibody pull-down and by yeast two-hybrid experiments that ALAS2 binds to SUCLA2 (22).…”

“…SUCLA2 Affinity Chromatography of ALAS2-Because the two exon 11 point mutations caused XLSA but did not significantly alter enzyme kinetics or stability, the possibility was explored that they disrupted the known interaction between ALAS2 and the beta subunit of the ATP-utilizing isozyme of succinyl-CoA synthetase (SUCLA2) (22). The MBP-SUCLA2 fusion protein was used as an affinity ligand to bind FPLCpurified wild type or mutant ALAS2.…”

Mitochondria in hematopoiesis and hematological diseases

“…In some families, the ALAS2 gene mutation either decreases the processing or the stability of the enzyme, leading to reduction of its level (Furuyama et al, 1997), or abrogates its interaction with protein partners (Furuyama and Sassa, 2000), thus rendering patients resistant to pyridoxine supplementation (Astner et al, 2005). Animal models for XLSA have been developed in mice and zebrafish by disruption of ALAS2 gene homolog (Nakajima et al, 1999;Yamamoto and Nakajima, 2000).…”

Expression of Two Succinyl-CoA Synthetases with Different Nucleotide Specificities in Mammalian Tissues

“…The switch is accompanied by the development of mitochondria, and the amount of A-SCS strongly increases along with other citric acid cycle enzymes (23). However, the finding in a yeast two-hybrid screen that A-SCS is an interacting protein with aminolevulinate synthase suggests that A-SCS can also support anabolic reactions (21).…”