title: The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis.
abstract: The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-aminolevulinate synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents approximately 35-45% of total ALAS2 mRNA while the newly identified transcripts together represent approximately 15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, SA 5005, Adelaide, Australia. timothy.cox@adelaide.edu.au
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Cox
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Sadlon
authors.forename: Quenten P
authors.initials: QP
authors.lastname: Schwarz
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Matthews
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Wise
authors.forename: Liza L
authors.initials: LL
authors.lastname: Cox
authors.forename: Sylvia S
authors.initials: SS
authors.lastname: Bottomley
authors.forename: Brian K
authors.initials: BK
authors.lastname: May
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002384:Catalysis; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005091:Exons; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006418:Heme / Q000096:biosynthesis*; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013389:Succinate-CoA Ligases / Q000737:chemistry; D020798:Two-Hybrid System Techniques
chemical_list: D003062:Codon; D006454:Hemoglobins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D006418:Heme; D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human; D012260:Ribonucleases; D013389:Succinate-CoA Ligases