The 23-megabase genome of Plasmodium falciparum, the causative agent of severe 
human malaria, contains ∼5300 genes, most of unknown function or lacking 
homologs in other organisms. Identification of these gene functions will help in 
the discovery of novel targets for the development of antimalarial drugs and 
vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning 
and expressing these genes in heterologous systems for functional analysis. The 
large repertoire of genetic tools available for Saccharomyces cerevisiae makes 
this yeast an ideal system for large scale functional complementation analyses 
of parasite genes. Here, we report the construction of a cDNA library from P. 
knowlesi, which has a lower A+T content compared with P. falciparum. This 
library was applied in a yeast complementation assay to identify malaria genes 
involved in the decarboxylation of phosphatidylserine. Transformation of a 
psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, 
with the P. knowlesi library led to identification of a new parasite 
phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine 
decarboxylase enzymes from other eukaryotes that are tightly associated with 
membranes, the PkPSD enzyme expressed in yeast was equally distributed between 
membrane and soluble fractions. In vitro studies reveal that truncated forms of 
PkPSD are soluble and undergo auto-endoproteolytic maturation in a 
phosphatidylserine-dependent reaction that is inhibited by other anionic 
phospholipids. This study defines a new system for probing the function of 
Plasmodium genes by library-based genetic complementation and its usefulness in 
revealing new biochemical properties of encoded proteins.