ABSTRACT
Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Heme/metabolism , Molecular Chaperones/metabolism , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Heme/chemistry , Humans , Mice , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Sequence Data , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Protein Binding , Sequence AlignmentABSTRACT
Maturation of adult (α2ß2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-ß) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-ß (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αß (RAW and A549) or that we transfected to express the globins. We found the following: (i) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbß or Hb-γ) and that hsp90 does not associate with mature Hb. (ii) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. (iii) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-ß, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis.
