An experimental tool to study exportin-1-dependent nuclear export in Plasmodium falciparum

Abstract text

Severe human malaria is caused by Plasmodium falciparum. In the clinically relevant blood stage of infection, P. falciparum proliferates through an unusual cell cycle mode called schizogony. During schizogony, the parasite"s nuclei multiply several times and a multinucleated cell is formed before daughter merozoites emerge. To facilitate rapid parasite proliferation, the nuclei multiply asynchronously despite residing in a shared cytoplasm. Moreover, the DNA replication fork protein P. falciparum PCNA1 accumulates only in those nuclei that replicate their DNA. One possibility to achieve this heterogenous nuclear accumulation is regulated nucleocytoplasmic transport. Especially regulated nuclear export may allow for protein accumulation in individual nuclei as nuclear export receptors bind their cargo in the confinements of the nucleus. In human cells, the nuclear export receptor exportin-1 transports roughly 1000 cargo proteins and has also been implicated in the export of the human PCNA1 homologue. Exportin-1-dependent nuclear export can be studied with the inhibitor leptomycin B (LMB), which covalently binds to a cysteine residue in the cargo binding pocked of exportin-1. However, P. falciparum is refractory to LMB inhibition as this cysteine is not conserved and a potential role for exportin-1 in the nuclear export of PCNA1 remains elusive. To develop an experimental tool, we genetically engineered P. falciparum by mutating isoleucine 637 of exportin-1 to cysteine (I637C). This rendered P. falciparum sensitive to LMB (EC₅₀ ~185 nM). Currently, we are characterizing the I637C mutant and exportin-1-dependent nuclear export using synthetic and predicted canonical cargo proteins. In parallel, we fused predicted nuclear import and export signals of P. falciparum PCNA1 to GFP. The localization of these constructs suggests that P. falciparum PCNA1 harbors at least one functional nuclear export signal. Together, this work will provide an experimental tool to study exportin-1-dependent nuclear export in P. falciparum. Investigating the nucleocytoplasmic transport will also reveal its relevance for heterogeneous nuclear accumulation of P. falciparum PCNA1. A better understanding of the fundamental biology of P. falciparum may also pave the way for new intervention strategies to curb malaria.