A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology
Figure 1
Identification of the ApiAP2 protein PfSIP2 as the SPE2-interacting protein.
(A) Gel shift assay to test the course of affinity purification. Lane 1: probe only; lane 2: nuclear extract (input); lanes 3–5: supernatants after incubation of nuclear extract with beads carrying single-stranded DNA (3), SPE2 (4) or mutated SPE2M (5); lanes 7–10: proteins eluted from SPE2-loaded beads (SPE2 E). Lanes 11 and 12: A second eluate (SPE2 E2) and proteins bound to mutated SPE2M (SPE2mut E) contain no SPE2-binding activity. Faster migrating bands are probably due to degradation of PfSIP2-N during affinity purification. (B) Schematic representation of the ApiAP2 protein PfSIP2 encoded by PFF0200c and recombinant epitope-tagged PfSIP2 proteins expressed in either P. falciparum (SIP2-Ty; SIP2-N-HA; SIP2-N-Ty) or E. coli (SIP2-N-HIS_A; SIP2-N-HIS_B). Dark and light blue boxes indicated AP2 domains 1 and 2. (C) Size estimation of the endogenous SPE2-binding activity in parasite nuclear extracts by UV-crosslinking. The arrow highlights the size of the crosslinked DNA-protein complex (lanes 2 and 4). Complex formation is competed by a 25-fold molar excess of SPE2 (lane 3) but not by mutated SPE2M (lane 4). The DNA-protein complex is not observed in absence of radiolabeled SPE2 (lane 1) or without prior UV-crosslinking (lane 5). (D) Gel shift assay to confirm the identity of PFF0200c as the SPE2-binding protein. Lane 1: probe only; lanes 2–5: 3D7 nuclear extract; lanes 6–9: nuclear extract of 3D7/SIP2-N-HA over-expressing SIP2-N-HA (aa 1–387); lane 10: untransformed E. coli control lysate; lanes 11–14: lysate of E. coli expressing recombinant SIP2-N-HIS_A (aa 1–387); lanes 15–18: lysate of E. coli expressing recombinant SIP2-N-HIS_B (aa 171–387). Lanes 3, 7, 12 and 16: 100-fold molar excess of unlabeled SPE2 competitor. Lanes 4, 8, 13 and 17: 100-fold molar excess of mutated competitor SPE2M. Lanes 5, 9, 14 and 18: EMSA supershift in presence of anti-HA or anti-6×HIS antibodies. (E) Anti-Ty Western blot of 3D7/SIP2-Ty nuclear extracts prepared at five consecutive timepoints during the intra-erythrocytic developmental cycle (IDC). Expression of full-length PfSIP2-Ty and subsequent processing occur during schizogony. The bottom panel shows the presence of PfSIP2-N by EMSA in schizont extracts. Protein extracted from equal numbers of nuclei were used in each lane. hpi: hours post-invasion. (F) Pull-down of full-length PfSIP2-Ty and PfSIP2-N-Ty based on affinity to SPE2. PfSIP2-N-Ty binds efficiently to immobilized SPE2 whereas neither full-length PfSIP2-Ty nor the C-terminal processed fragment PfSIP2-C-Ty interact with SPE2. None of the proteins bound to mutated SPE2M DNA. SN: supernatant.
