ABSTRACT
IMPORTANCE: Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.
Subject(s)
Plasmodium , Protozoan Proteins , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Plasmodium/genetics , Protein Transport , Erythrocytes/parasitology , Endoplasmic Reticulum/metabolism , Plasmodium falciparum/metabolismABSTRACT
Proteases of the malaria parasite Plasmodium falciparum have long been investigated as drug targets. The P. falciparum genome encodes 10 aspartic proteases called plasmepsins, which are involved in diverse cellular processes. Most have been studied extensively but the functions of plasmepsins IX and X (PMIX and PMX) were unknown. Here we show that PMIX is essential for erythrocyte invasion, acting on rhoptry secretory organelle biogenesis. In contrast, PMX is essential for both egress and invasion, controlling maturation of the subtilisin-like serine protease SUB1 in exoneme secretory vesicles. We have identified compounds with potent antimalarial activity targeting PMX, including a compound known to have oral efficacy in a mouse model of malaria.
Subject(s)
Antimalarials/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Malaria, Falciparum/drug therapy , Plasmodium falciparum/enzymology , Administration, Oral , Animals , Antimalarials/administration & dosage , Antimalarials/chemistry , Antimalarials/therapeutic use , Disease Models, Animal , Erythrocytes/parasitology , Mice , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/pathogenicity , Protozoan Proteins/metabolism , Subtilisins/metabolismABSTRACT
The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in N(6)-threonylcarbamoyl adenosine tRNA modification, transcriptional regulation, and telomere homeostasis. These functions are performed in complex with a conserved core of protein binding partners. Herein we describe the localization, essentiality, and protein-protein interactions for Kae1 in the human malaria parasite Plasmodium falciparum. We found that the parasite expresses one Kae1 protein in the cytoplasm and a second protein in the apicoplast, a chloroplast remnant organelle involved in fatty acid, heme, and isoprenoid biosynthesis. To analyze the protein interaction networks for both Kae1 pathways, we developed a new proteomic cross-validation approach. This strategy compares immunoprecipitation-MS data sets across different cellular compartments to enrich for biologically relevant protein interactions. Our results show that cytoplasmic Kae1 forms a complex with Bud32 and Cgi121 as in other organisms, whereas apicoplast Kae1 makes novel interactions with multiple proteins in the apicoplast. Quantitative RT-PCR and immunoprecipitation studies indicate that apicoplast Kae1 and its partners interact specifically with the apicoplast ribosomes and with proteins involved in ribosome function. Together, these data indicate an expanded, apicoplast-specific role for Kae1 in the parasite.
Subject(s)
Apicoplasts/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Ribosomes/metabolism , Alleles , Amino Acid Sequence , Animals , Cell Compartmentation , Cluster Analysis , Conserved Sequence , Cytosol/metabolism , DNA, Protozoan/metabolism , Humans , Life Cycle Stages , Mass Spectrometry , Molecular Sequence Annotation , Molecular Sequence Data , Nucleic Acids/metabolism , Parasites/growth & development , Phylogeny , Plasmodium falciparum/growth & development , Protein Binding , Protein Interaction Maps , Protein Transport , Protozoan Proteins/chemistry , RNA, Ribosomal/metabolism , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.
