Functional analysis of exported Plasmodium falciparum proteins by specific conditional regulation of protein transport
Final Report Abstract
Mouse dihydrofolate reductase (mDHFR) folds tightly into its tertiary structure in the presence of folate analogues, even if mDHFR is expressed as part of a chimeric fusion protein. Using the mDHFR system it has been shown that transport of soluble exported P. falciparum proteins require a protein-unfolding step within the parasitophorous vacuole and the addition of a folate analogue blocked export of a chimeric PEXEL-containing mDHFR-GFP reporter. Thus, this system provides a method with which the transport of a protein to its site-of-action can be specifically blocked. Further it allows generating a regulated protein export systems to specifically control the trafficking of essential proteins to the host cell, and their functional localisation. As trafficking of the protein of interest is inhibited only upon addition of a folate analogue, but otherwise takes place normally, this system is used to characterise the function of essential parasite-encoded exported heat shock 40 proteins (PfHsp40s) whose functions could not be addressed with current gene inactivating strategies. Although this system does require further optimisation, which could not be completed, initial results suggests that this system can be used for the study of phenotypic changes caused by the conditional regulation of protein transport individual candidate proteins.
