Multiple autophosphorylation is essential for the formation of the active and stable homodimer of heme-regulated eIF2alpha kinase.

In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2alpha kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme.

Two heme-binding domains of heme-regulated eukaryotic initiation factor-2alpha kinase. N terminus and kinase insertion.

In heme deficiency, protein synthesis in reticulocytes is inhibited by activation of heme-regulated alpha-subunit of eukaryotic initiation factor-2alpha (eIF-2alpha) kinase (HRI). Previous studies indicate that HRI contains two distinct heme-binding sites per HRI monomer. To study the role of the N terminus in the heme regulation of HRI, two N-terminally truncated mutants, Met2 and Met3 (deletion of the first 103 and 130 amino acids, respectively), were prepared. Met2 and Met3 underwent autophosphorylation and phosphorylated eIF-2alpha with a specific activity of approximately 50% of that of the wild type HRI. These mutants were significantly less sensitive to heme regulation both in vivo and in vitro. In addition, the heme contents of purified Met2 and Met3 HRI were less than 5% of that of the wild type HRI. These results indicated that the N terminus was important but was not the only domain involved in the heme-binding and heme regulation of HRI. Heme binding of the individual HRI domains showed that both N terminus and kinase insertion were able to bind hemin, whereas the C terminus and the catalytic domains were not. Thus, both the N terminus and the kinase insertion, which are unique to HRI, are involved in the heme binding and the heme regulation of HRI.

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.

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in the bacteria grown in the cell culture medium have been induced during intracellular growth in macrophages. In contrast, approximately 50 proteins that were expressed during growth in cell culture medium were completely repressed during intracellular growth. The level of expression of 19 proteins increases while that of 54 proteins decreases during intracellular growth. To understand these results, the protein synthesis pattern of B. abortus during intracellular growth was compared with those during other stress conditions. Under each stress condition studied, several new proteins were induced that were not present during regular growth conditions. Comparison of the protein synthesis pattern of B. abortus during intracellular growth with those obtained under various stress conditions has indicated that the response to intracellular growth was not just a simple sum of stress conditions studied so far.

Composition and structure of the venus atmosphere: results from pioneer venus.

Results from the Pioneer Venus sounder probe neutral mass spectrometer indicate that there is no difference in the isotopic ratios of carbon and oxygen between Venus and Earth to within +/- 5 percent. The mixing ratio of nitrogen is 3.5(+3)(-2) percent with an isotopic ratio within 20 percent of that of Earth. The ratio of argon-36 to argon-40 is 85 percent, and the ratio of argon-38 to argon-36 is 20 percent. The mixing ratios of argon-36 and argon-40 are approximately 40 and 50 parts per million, respectively, with an error of about a factor of 2 (mainly toward a lesser amount) resulting from uncertainty in the response of the ion pump to rare gases. Hydrogen chloride cannot account for more than a few percent of the 36 mass peak, and therefore the large excess of primordial argon is a reasonable conclusion. The ratio of neon-20 to argon-36 of 0.5 +/- 0.3 is definitely terrestrial in character rather than solar. These results indicate that there is a large excess of all primordial noble gases on Venus relative to Earth. There appears to be a considerably higher abundance of sulfur compounds below 20 kilometers than in or above the main cloud layer. The 32 and 60 mass peaks show a sharp increase below 22 kilometers, indicating the possible production of sulfur and carbon oxysulfide (COS) at the expense of sulfur dioxide.

Venus lower atmospheric composition: preliminary results from pioneer venus.

Initial examination of data from the neutral mass spectrometer on the Pioneer Venus sounder probe indicates that the abundances of argon-36, argon-38, and neon-20 in the Venus atmosphere are much higher than those of the corresponding gases in Earth's atmosphere, although the abundance of radiogenic argon-40 is apparently similar for both planets. The lower atmosphere of Venus includes significant concentrations of various gaseous sulfur compounds. The inlet leak to the mass spectrometer was temporarily blocked by an apparently liquid component of the Venus clouds during passage through the dense cloud layer. Analysis of gases released during the evaporation of the droplets shows the presence of water vapor to some compound or compounds of sulfur.