Plasmodium falciparum: Funktionelle Analyse von Proteinen des sekretorischen Transportweges in transfizierten Zellen

Plasmodium falciparum is an intracellular parasite of red blood cells. The parasite exports various proteins to numerous destinations inside of its plasma membrane and beyond into the host erythrocyte. These secreted proteins are directly related to the severe clinical symptoms of malaria tropica. The export of most parasite proteins is inhibited in the presence of brefeldin A (BFA). The conserved sec7 domain of Arf-Gef (ADP-ribosylation factor - guanine nucleotide exchange factor) is the main target of BFA. Arf-Gef proteins are necessary to activate Arf and consequently for the formation of COP I coated transport vesicles. Here it is shown by double cross-over gene replacement experiments with P. falciparum that a point mutation within the sec7 domain is sufficient to confer BFA-resistance to parasites. Complementation studies with the yeast S. cerevisiae in which the gea1 sec7 domain was replaced by the P. falciparum sec7 domain including the mutation revealed an intermediate growth phenotype. This provides evidence that P. falciparum Arf-Gef functions as a GDT-GTP-exchange protein in ER-/Golgi transport processes. The sec7 region of the arf gef gene encodes an unusual long insertion sequence. In silico modelling algorithms as well as heterologous expression in yeast cells were used here to examine the significance of this insertion sequence. Parasite proteins that are transported into intra-parasitic compartments like the apical complex or the apicoplast in many cases contain amino-terminal ER signal sequences. Many proteins destined for the host cell compartment contain internal hydrophobic regions which are putative ER-signals. The internal hydrophobic region of Gbp130 (glycophorine binding protein) and truncated versions thereof were functionally analysed as to their ER-signal potential in S. cerevisiae. The P. falciparum Exp-1 signal sequence was used as a positive control. None of the plasmodial sequences could restore ER-translocation in vivo possibly due to parasite specific transport/ translocation processes that could not be reconstituted in yeast cells. Two aspects of secretory processes in P. falciparum were investigated in this thesis. For an understanding of protein export pathways and mechanisms it is necessary to identify and characterize more mediator molecules involved in parasite secretion.