A nuclear targeting system in Plasmodium falciparum.
BACKGROUND
The distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the P. falciparum nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the P. falciparum nucleus using an exogenous nuclear localization sequence is reported here.


METHODS
To develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for P. falciparum nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy.


RESULTS
The nuclear localization sequence of Gal4 is functional in P. falciparum. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization.


CONCLUSIONS
The Gal4 fusion technique enables specific transport of a protein of choice into the P. falciparum nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and P. falciparum. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in P. falciparum.
Wittayacom
Kanjana
K
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Uthaipibull
Chairat
C
Kumpornsin
Krittikorn
K
Tinikul
Ruchanok
R
Kochakarn
Theerarat
T
Songprakhon
Pucharee
P
Chookajorn
Thanat
T
D021581:Active Transport, Cell Nucleus; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000235:genetics / Q000502:physiology*; D018087:Plastids / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
C064255:GAL4 (1-147) protein, S cerevisiae; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins
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