Colocalization analysis of PfCCT construct in CHO-MT58 cells. Confocal immunofluorescence images of cells transfected with (A) the full length PfCCT, (B) the second repeat unit C2M2 construct, (C) the lysine-rich loop truncated C2M2ΔK construct, (D) the membrane-binding domain truncated C2 construct, (E) C2ΔK and (F) SV40 NLS-tagged C2M2 construct. (G–L) Confocal images of cells transfected with PfCCT constructs and treated with PLC. White lines indicate 50 μm scalebars. (K) Nuclear overlap coefficients were calculated with Fiji. The number of cells used for the calculation are indicated in the columns. *p < 0.001. To elucidate the impact of the proposed regions on the nuclear localization, confocal microscopy-based colocalization analysis was applied. Both the full length PfCCT and the second repeat unit construct, C2M2 has a mixed nuclear-cytoplasmic localization in CHO-MT58 cells (A,B). However, the lysine-rich loop truncated C2M2ΔK construct showed a predominantly cytosolic localization, which indicates the nuclear localization signal role of this insertion (Fig. 3C). Furthermore, the membrane-binding domain truncated second repeat unit construct, C2 also showed a decreased nuclear localization compared to the C2M2 construct and the full length PfCCT protein (D,H,M). We also investigated the consequences of a double truncated C2ΔK construct, which showed a decreased nuclear accumulation similar to the C2 and C2M2ΔK constructs (E,K).
Izrael R, Marton L, Nagy GN, Pálinkás HL, Kucsma N, Vértessy BG. Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme. Sci Rep. 2020 Nov 12;10(1):19739.