Heme-regulated eIF-2alpha kinase purifies as a hemoprotein.
The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme-regulated eIF-2alpha kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF-2alpha kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent Ki of 0.5 microM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of heme-binding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.
Chefalo
P J
PJ
Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts 02139, USA.
Oh
J
J
Rafie-Kolpin
M
M
Kan
B
B
Chen
J J
JJ
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods; D019281:Dimerization; D015536:Down-Regulation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005914:Globins / Q000096:biosynthesis; D006418:Heme / Q000096:biosynthesis; D006420:Hemeproteins / Q000737:chemistry*; D006427:Hemin / Q000378:metabolism / Q000494:pharmacology*; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012156:Reticulocytes / Q000201:enzymology*; D013053:Spectrophotometry; D019892:eIF-2 Kinase / Q000737:chemistry*
D004791:Enzyme Inhibitors; D006420:Hemeproteins; D011994:Recombinant Proteins; D006418:Heme; D006427:Hemin; D005914:Globins; D019892:eIF-2 Kinase