Identification of trafficking determinants needed for targeting of apical organelle proteins in Plasmodium falciparum
Date
2014-08-25
Authors
Wepener, Melanie
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
To invade host erythrocytes, the malaria parasite, Plasmodium falciparum, produces
invasion proteins, which are translocated through the secretory pathway by an N-terminal
signal peptide. Additional motifs allow the proteins to be transported to the secretory
organelles, the rhoptries and micronemes. Proteins of the Duffy Binding-Like Erythrocyte
Binding Protein (DBL-EBP) family possess a C-terminal cysteine-rich domain, proposed
to be the motif necessary for targeting. To investigate this hypothesis, mini-gene constructs
were created from two DBL-EBPs, MAEBL and JESEBL by overlap extension PCR, and
used to transfect 3D7 P. falciparum parasites. It was anticipated that only chimaeric
proteins containing the domain would be correctly localized to the secretory organelles,
while those without the domain would remain within the cytoplasm. Two constructs were
produced for MAEBL, containing the signal peptide, transmembrane domain and
cytoplasmic tail, with the C-terminal cysteine-rich domain either present, M1 (CCys), or
absent, M2 ( CCys). These were cloned into the pARL-GFP plasmid that had been
modified by replacing the crt promoter with the ama-1 promoter for stage-specific
expression. A third construct contained the JESEBL signal peptide and C-terminal
cysteine-rich domain, J (CCys), which was cloned into the pARL-mCherry/ama-1
promoter plasmid. Successfully transfected parasites were visible 25-30 days posttransfection
and the presence of the episomal plasmids was verified by PCR. Microscopic
analysis of live parasites revealed diffuse fluorescence in the cytoplasmic regions for
control transfectants carrying the empty pARL2-GFP and pARL-mCherry vectors, as
expected. The chimaeric M2 ( CCys) protein showed a similar pattern to the control GFP,
however, chimaeric M1 (CCys) was localized to the parasite apex, indicating secretory
organelle localization. These results showed that the C-terminal cysteine-rich domain is
sufficient and necessary for apical trafficking of MAEBL. The chimaeric J (CCys) protein
was localized throughout the cytoplasmic and apical regions, indicating partial trafficking
of the protein to the secretory organelles. This was further confirmed by co-localization
studies whereby EBA-175 was used as a micronemal marker. The JESEBL C-terminal
cysteine-rich domain is therefore sufficient but not completely adequate for correct protein
trafficking. Based on these data and other published studies, two hypothetical models are
proposed for the transport of MAEBL and JESEBL to the apical organelles.