Fig. 1. (A) Schematic representation of PfKarα showing different protein domains identified by Pfam. E-value cut-off used was 0.1, as domains with values less than 0.1 are considered as very significant [22]. All the domains had E-values much less than 0.1, which are indicated in brackets; respective amino acid positions are also indicated. IBB, importin β-binding domain; ARM, Armadillo/β-catenin-like repeat. (B) Amino acid sequence alignment of PfKarα IBB domain region with that of six homologs from Caenorhabditis elegans (Q19969), Homo sapiens [AJ303086 (karα2) and NP_036448 (karα6d)], Xenopus laevis (L36340), Drosophila melanogaster (AF074957), and Mus musculus (Q60960). Amino acids that are identical in at least five of seven species (>70%) are shown in red, amino acids that are similar in at least five of seven species (>70%) or to those shown in red, are shown in blue. Residues that have been shown by crystallographic studies to be involved in binding with the karyopherin β are indicated by solid bar. (C) Schematic representation of PfKarβ showing different protein domains, E-values obtained for the domains are given in brackets, respective amino acid positions are also indicated. Overlapping motifs among other karyopherin β members of respective Pfam-B family proteins are indicated below. IBN-NT, importin β N-terminal domain. (D) Amino acid sequence alignment of HEAT region of Pfam-B 821 domain of PfKarβ with homologous region of other karyopherin β members of the protein family. HEAT domains identified in humans by crystallography are marked with solid bar. (E) Amino acid sequence alignment of Pfam-B 6210 domain of PfKarβ with homologous region of karyopherin β of other organisms. The region identified as HEAT or HEAT-like domains in humans and yeast is indicated by solid bar. (F) Amino acid sequence alignment of HEAT region of Pfam-B 1263 domain of PfKarβ with homologous region of other karyopherin β members of the protein family. Region identified as HEAT or HEAT-like domains in human and yeast are indicated by solid bar. The color convention used for sequence alignments in panels D–F is same as in panel B. The sequences used for alignment in panels D–F are: H. sapiens [NP_00261 (karβ2) U72761 (karβ3)], Saccharomyces cerevisiae [P38217 (karβ2), P32337 (karβ3), p40069 (karβ4)], Schizosaccharomyces pombe [NP_588502, NP_596729 (karβ4)], D. melanogaster [NM_079502 (karβ3)], and M. musculus (AF294327).

Fig. 2. (A) PCR amplification of PfKarα (lane 1) and PfKarβ (lane 2) from genomic DNA using respective terminal primers. (B) and (C) RT–PCR analyses of PfKarα (B; lane 1, RT+, lane 2, RT−) and PfKarβ (C) from total RNA. Total RNA was isolated from asynchronous blood stage parasite culture using RNAeasy kit (Qiagen) and treated with DNase; cDNA was prepared using reverse transcription kit (GIBCO BRL) following manufacturer’s recommendations. (D) and (E) Northern blot analyses to confirm stage-specific transcription of PfKarα (D) and PfKarβ (E). Total RNA was isolated from synchronized parasite cultures at ring (lane 1), trophozoite (lane 2), and schizont (lane 3) stage, 2 μg of each sample was separated on a denaturing 1.2% agarose gel, Northern blotted, and hybridized with labeled PfKarα probe (D), equal loading of RNA in all the wells was confirmed by ethidium bromide staining of rRNAs in the gels (lower panel); the membrane was deprobed and hybridized with the PfKarβ probe (E). (F–I) Expression and binding of PfKarα and PfKarβ. The PfKarβ gene was cloned into pQE30 vector (Qiagen); recombinant protein was expressed in Escherichia coli with His-tag and purified using Ni-NTA agarose beads (Qiagen) and Q-Sepharose (Pharmacia Biotech). PfKarα was cloned in pIVEX2.3 (Roche Biochem) and ³⁵S-labeled protein was expressed without His-tag using in vitro translation kit (Roche Biochem). (F) Coomassie blue-stained SDS–PAGE and (G) Western blot (using anti-His antibodies) of PfKarβ protein. (H) Western blot of blood stage parasite lysate using anti-PfKarβ antibodies. (I) Autoradiogram of SDS–PAGE showing labeled PfKarα protein. (J) Solution-binding assay of PfKarα and PfKarβ. About 2 μg of purified PfKarβ recombinant protein was allowed to bind with Ni-NTA agarose beads for 2 h in TB-T (20 mM HEPES, 110 mM potassium acetate, 1 mM EGTA, pH 7.3, 2 mM DTT, 0.1% Tween 20). The beads were washed thoroughly and incubated with labeled PfKarα in TB-T for 1 h at 4 °C. After thorough washing, the bound proteins were analyzed by SDS–PAGE (lane 1). Gels were exposed to X-ray films for autoradiography. His-tagged PfKarβ was exchanged with His-tagged PfMSP-1₁₉ in the assay for the negative control (lane 2).