New insights into PEXEL-mediated protein export in Plasmodium falciparum: The role of N-terminal acetylation

Abstract

The apicomplexan parasite Plasmodium falciparum is the causative agent of the most severe form of malaria with more than 600 000 death cases per year, mainly among young children and pregnant women. The development of the parasite within human erythrocytes is accompanied by extensive remodelling of the host cells. The establishment of nutrient acquisition pathways, the genesis of membranous structures within the red blood cell (RBC) cytosol and the formation of a cytoadherence complex on the surface of the host cell represent important examples of these modifications that contribute both to parasite survival and virulence. For this purpose P. falciparum exports hundreds of effector proteins into the RBC with the majority of them bearing a characteristic pentameric sequence termed Plasmodium export element (PEXEL), which is cleaved in the parasite after the third and N-terminally acetylated at the fourth amino acid position. To gain a better understanding of this important export signal, a mutagenesis screen on the PEXEL motif (48RLLAQ52) of a GFP-tagged model protein (STEVOR1-80) was conducted in this study. The localization of the mutant proteins to different compartments of the parasitized erythrocyte was determined by fluorescence microscopy. In addition, mass spectrometric analysis of representative mutants confirmed a correlation between processing of the export motif and protein trafficking. In summary, amino acid replacements within position 1 (R48) and 3 (L50), representing the most conserved amino acids, had detrimental effects on PEXEL cleavage and protein export. In contrast, positions 2 (L49) and 5 (Q52) proved to be more permissive towards mutations and mostly maintained the wild-type export phenotype. Special attention was paid to position 4 (A51), which is acetylated after cleavage and appeared more restricted than originally assumed. One interesting finding was the deficiency of non-acetylated mutant proteins to be exported into the host cell indicating N-terminal acetylation as a prerequisite for proper targeting of PEXEL proteins. Consequently, the second part of this study focussed on the characterization of a putative N-acetyltransferase (PfNAT) that might be responsible for this allegedly important post-translational modification. The localization of the candidate transferase to the parasite endoplasmic reticulum, the compartment of PEXEL processing, was confirmed by single crossover integration of a GFP tag into the endogenous locus. Despite the use of two different approaches, the attempted deletion of the gene for loss-of-function studies was unsuccessful. However, a conditional downregulation of protein levels by ~50% was achieved using a destabilization domain, which facilitated further investigations.