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VOL. 72 (4), 611-627, 2006 HEME RESPONSIVENESS IN VITRO IS A COMMON...

we constructed the HRI Q4 mutant, in which the entire kinase insert
together with the N-terminal of HRI kinase subdomain V (11) was
replaced by the corresponding amino acid sequence of mouse GCN2
(5). All the HRI constructs in pRSETB vector (11) were efficiently
expressed in E. coli and the amounts of recombinant proteins, tested
by Western blot, were similar (data not shown).

To assess the kinase activity of the HRI mutants, an in vitro
kinase assay was performed in which bacterial extracts containing
these mutants were tested for phosphorylation of exogenously added
eIF2. Under these conditions, the extent of inhibition of eIF2a kinase
activity of the wild type and HRI mutants by heme was dependent
upon the amount of HRI used.

To determine the role of the HRMs in the heme regulation of
HRI we studied the effect of hemin on the eIF2a kinase activity
of three different HRI mutants, C409A/C550A, ?8, and ?8/C550A.
For all of the mutants, eIF2a kinase activity was similar to that of
the wild type HRI. Furthermore, the eIF2a kinase activity of HRI
mutants was also hemin-sensitive (Fig. 2A). Interestingly, the extent
of inhibition of HRI mutants by heme was similar to that of HRI wt
at all hemin concentrations tested (Fig. 2B). The concentration of
hemin required for 50% inhibition of eIF2a kinase activity was about
0.3-0.5 µM for wild type HRI, in good agreement with earlier studies
(3, 18), and 0.3 µM, 0.4 µM and 0.7 µM for C409A/C550A, ?8 and ?8/
C550A mutants, respectively. These results strongly suggest that the
entire HRM1 and the cysteine residue in HRM2 have no apparent
effect on the heme-responsiveness of HRI in vitro.

Similarly, to determine the role of both amino and carboxyl ends
in the heme regulation of HRI, the inhibition by heme of the eIF2a
kinase activity of the two indicated HRI deletion mutants, also
containing point mutations into the HRMs (?137/C409A/C550A and
138-580/C409A/C550A), were compared. In this case, the eIF2a kinase
activity of mutants was also inhibited by the same concentration
range of hemin (Fig. 2A), although we obtained slightly different
values in the hemin concentration required for their half-maximal
inhibition (Fig. 2B). These results suggest that neither N- nor C-
terminal domains are essential in the heme regulation of HRI.
Finally, the mutant HRI Q4, in which the HRI kinase insert had been

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