The P. falciparum eIF2α orthologue is phosphorylated in response to amino-acid starvation. A: Alignment of PfeIF2α with orthologous sequences from T. gondii (Tg), human (Hs), rice (Os) and E. cuniculi (Ec). Sequences surrounding the conserved regulatory serine, (P. falciparum numbering: M48 – K108) are shown. Residues that are identical in all sequences are highlighted in black, residues that are identical or similar are marked in grey. The arrow indicates the serine that is the target of eIF2α kinases. Open arrow heads (∨) indicate residues involved in contacting the kinase domain, asterisks (*) indicate conserved residues that protect the phosphorylation site from the activity of other kinases. B. Western blot analysis of PfeIF2α phosphorylation. A 3D7 parasite culture synchronized to the late ring stage was equally partitioned into individual cultures. Growth of the parasites was continued up to 5 hours at 37°C in either complete RPMI medium (CM) or in RPMI lacking amino acids (-AA). CM was added back to one amino acid-deprived culture, and re-incubated for an additional 45 minutes. Total lysates from the parasites were prepared for SDS-PAGE, followed by immunoblotting with antibodies against phosphorylated eIF2α (anti-phospho eIF2α) and the endoplasmic reticulum (ER) marker, BiP (anti-BiP), which served as the loading control.