The N-terminus of UIS2 binds eIF2α-P.

Abstract

<p>(A) PfeIF2α-P interacts with endogenous UIS2. In these experiments we used the codon-optimized eIF2α of <i>P</i>. <i>falciparum</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.ref007" target="_blank">7</a>] that shares 89% identity with its <i>P</i>. <i>berghei</i> ortholog. The immobilized GST-PfeIF2α was incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites in the presence or absence of Sal (50 μM) or GA (70 μM). The bound proteins were detected by immunoblot using antibodies against PP1, UIS2, phosphorylated eIF2α, and total eIF2α. Levels of PbeIF2α-P and total PbeIF2α were quantified by densitometry analysis. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s010" target="_blank">S8 Fig</a>: The mouse anti-PbPP1 antibody recognized the 35 kDa endogenous PP1 from the parasite lysates. (B) PfeIF2α<i>S59D</i> mutant protein interaction with UIS2. The immobilized GST-PfeIF2α wt, <i>S59A</i>, or <i>S59D</i> were incubated with the lysates of <i>P</i>. <i>berghei</i> blood stage parasites, with or without Sal, and UIS2 was detected by immunoblot. (C) Schematic representation of the PbUIS2 coding sequence. UIS2 contains a putative conserved phosphatase domain (PD, 535–1054 amino acids) and <i>Plasmodium</i> specific sequences at N- and C- terminus. (D) The N-terminus of PbUIS2 bound PfeIF2α-P. The PbUIS2 N-ter, PD, and C-ter were fused to GST-tag at their N-terminus and His-tag at their C-terminus, respectively. After 2-step affinity purification, the <i>E</i>. <i>coli</i> expressed fusion proteins were immobilized on glutathione sepharose 4B. After incubation with purified recombinant PfeIF2α-P, the sepharose was washed three times with high-salt NETN buffer (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 0.5% (v/v) Nonidet P-40). The retained proteins were detected by SDS-PAGE followed by coomassie brilliant blue staining. Lane 1,3,5: GST and His-tagged PbUIS2 N-ter, PD, and C-ter, respectively. Lane 2,4,6: proteins retained on the glutathione sepharose 4B after pull down assays with PfeIF2α-P. Lane 7, PfeIF2α-P control. The pulled down 38 kDa protein in lane 2 was analyzed by mass spectrometry and identified as PfeIF2α-P. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005370#ppat.1005370.s011" target="_blank">S9 Fig</a>. (E) Co-immunoprecipitation (IP) of endogenous complex between UIS2 and eIF2α-P. Whole cell extracts from <i>P</i>. <i>berghei</i> blood stage parasites were subjected to immunoprecipitation with anti-eIF2α-P or anti-UIS2 antibodies followed by immunoblot analysis.</p