Plasmodium falciparum is responsible for the deadliest form of human malaria. The underlying gene control mechanisms have been an active area of research for years owing to the potential for drug and vaccine development [
1]. The parasite has a membrane-bound nucleus with a haploid genome of 23-Megabases encoded in 14 chromosomes [
2]. The parasite undergoes mitotic division in red blood cells to generate 8-24 new nuclei in approximately 48 hours. Its chromosomes contain telomeric and putative centromeric regions [
3‐
5]. Despite the paucity of
P. falciparum transcription factors with clear homologues in other species [
6], stage-specific gene expression patterns were observed by microarray studies suggestive of transcriptional control [
7,
8]. The role of epigenetic gene regulation in the parasite is becoming clearer, with revised gene annotation revealing components of histone-modifying enzymes and modified histone readers [
9]. Furthermore, nuclear compartmentalization has been suggested to play an important role in gene regulation by forming transcriptionally active and silenced areas in the nucleus [
10]. Gene control mechanisms in malaria parasites are a fascinating subject, but there are few molecular toolkits available to study the nucleus of
P. falciparum. The ability to specifically transport a protein of choice to the nucleus is a valuable tool in many model organisms. This technique could be used to dissect the molecular basis of
P. falciparum nuclear function by targeting of exogenous proteins into the nucleus.
Malaria parasite uses short peptide sequences to determine the destination of newly-synthesized proteins. For example, the PEXEL/VTS sequence is responsible for targeting malarial proteins to the red blood cell membrane [
11,
12]. Proteins are also transported into the apicoplast using a bipartite signal sequence to facilitate apicoplast targeting and membrane translocation [
13,
14]. These findings open new opportunities for developing molecular toolkits for targeting a protein of choice to various malaria organelles. An example of this organelle targeting approach is the use of Pfhsp60 N-terminal sequence to send proteins to the mitochondrion [
15]. To address the lack of a nuclear targeting technique in
P. falciparum, a similar approach was adopted by utilizing the yeast transcription factor Gal4 as a nuclear carrier protein. The Gal4 protein (Gal4p) is one of the first well-studied DNA-binding transcription factors in the budding yeast
Saccharomyces cerevisiae[
16]. The protein is actively imported into the yeast nucleus and binds to
GAL upstream activation sequence [
17,
18]. A nuclear localization signal (NLS) at its N-terminus is a protein region recognized by the yeast nuclear transport machinery, and is necessary for targeting Gal4p into the nucleus [
16]. Since its nuclear localization sequence has been accurately delimited [
16], the potential use of Gal4p as a nuclear targeting system in
P. falciparum was tested. Here, the nuclear localization signal of Gal4p was shown to be compatible with
P. falciparum and can effectively transport a protein of choice into the nucleus. Truncated versions of Gal4p were analysed by confocal microscopy to narrow down the minimal nuclear localization region functioning in the malaria parasite. The Gal4-nuclear targeting system was also shown to be compatible with different
P. falciparum strains.