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

Mg(OAc)2, 0.25 mg/ml BSA, 50 µM ATP) containing purified rabbit
reticulocyte eIF2 (0.5 µg) and 3 µCi of [?-32P] ATP (3000 Ci/mmol).
When PKR or GCN2 was assayed the reaction mixtures also included

1 µg/ml poly(I)·poly(C) dsRNA or 2.5 µg/ml (0.65 nM) Sindbis virus
RNA, respectively. Phosphorylation of casein (Sigma) by highly

purified casein kinase II (CKII) from rat liver was carried out as

described (22). Briefly, purified CKII (0.01 U/ml) was incubated for

10 min at 30º C with 66 µg/ml casein as substrate and 3 µCi of
[?-32P] ATP (3000 Ci/mmol) in a final volume of 30 µl containing
20 mM Tris-HCl (pH 7.2), 8 mM MgCl2, 0.5 mM EGTA, 0.5 mM
EDTA, 1 mM dithiothreitol and 50 µM ATP. ?PKC kinase assay was
as for eIF2a kinases, except for the substitution of Mg(OAc)2 by
MgCl2 and eIF2a by 3 µg of myelin basic protein (MBP) as substrate.
Incubations were terminated by the addition of SDS sample buffer

and phosphoproteins were analyzed by both SDS-PAGE (23) on a 10

or 12.5% polyacrylamide gel (28.5:1 (w/w) acrylamide/bisacrylamide)

and autoradiography using Agfa Curix RP2 film and an Amersham-

Pharmacia intensifying screen. The areas corresponding to the

phosphorylated a-subunit of eIF2 were scanned at 633 nm in a
computing 300 A densitometer (Molecular Dynamics, Inc.).

RESULTS

All known HRIs contain two putative heme regulatory motifs
(HRM): ACPYVM and RCPVQA, which are located within the
catalytic domain [(11) and Fig. 1B] and are not present in other
eIF2a kinases. To date, the significance of these motifs in the heme
regulation of HRI is unknown.

To understand the regulation mechanism of HRI by heme, we
designed and constructed a series of mutants (Fig. 1A), including
point mutations of the two invariant cysteine residues in the HRMs
(HRI C409A/C550A) alone or together with either a large N-terminal
deletion (HRI ?137/C409A/C550A) or deletions that remove both the
N-terminal and the C-terminal domains (HRI 138-580/C409A/C55A).
The HRI ?8 mutant contains a deletion of amino acids 406-413 that
removes the entire HRM1. The HRI ?8/C550A mutant also contains
a single point mutation of conserved cysteine in HRM2. In addition,

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