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Abstract: The poor expression of Plasmodium falciparum proteins in heterologous systems and the difficulty in obtaining sufficient material directly from the parasite have limited the experimental characterization of many of the approximately 5200 proteins encoded by this organism. To improve the expression of P. falciparum proteins in the yeast Saccharomyces cerevisiae, we selected yeast ura3 mutants that acquired the ability to utilize the P. falciparum orthologue (PfOMPDC) of URA3 to grow on media lacking uracil. Two of these mutant strains, BY#29 and PJ#17, expressed up to 100-fold more of four P. falciparum proteins as a result of mutations in either HRP1 or KAP104, respectively. These mutations, as well as a temperature-sensitive rna15 mutation, likely decrease the efficiency of mRNA 3′ end formation and produce longer mRNAs of P. falciparum genes. These yeast strains may be useful for the analysis and purification of P. falciparum proteins. [Copyright &y& Elsevier]

Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein–protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen. [ABSTRACT FROM AUTHOR]

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1-TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) subcomplexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNAiMet binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of MettRNAiMet and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex. [ABSTRACT FROM AUTHOR]

Summary Centromeres are the chromosomal regions promoting kinetochore assembly for chromosome segregation. In many eukaryotes, the centromere consists of up to mega base pairs of DNA. On such “regional centromeres,” kinetochore assembly is mainly defined by epigenetic regulation [ 1 ]. By contrast, a clade of budding yeasts ( Saccharomycetaceae ) has a “point centromere” of 120–200 base pairs of DNA, on which kinetochore assembly is defined by the consensus DNA sequence [ 2, 3 ]. During evolution, budding yeasts acquired point centromeres, which replaced ancestral, regional centromeres [ 4 ]. All known point centromeres among different yeast species share common consensus DNA elements ( CDE s) [ 5, 6 ], implying that they evolved only once and stayed essentially unchanged throughout evolution. Here, we identify a yeast centromere that challenges this view: that of the budding yeast Naumovozyma castellii is the first unconventional point centromere with unique CDE s. The N. castellii centromere CDE s are essential for centromere function but have different DNA sequences from CDE s in other point centromeres. Gene order analyses around N. castellii centromeres indicate their unique, and separate, evolutionary origin. Nevertheless, they are still bound by the ortholog of the CBF3 complex, which recognizes CDE s in other point centromeres. The new type of point centromere originated prior to the divergence between N. castellii and its close relative Naumovozyma dairenensis and disseminated to all N. castellii chromosomes through extensive genome rearrangement. Thus, contrary to the conventional view, point centromeres can undergo rapid evolutionary changes. These findings give new insights into the evolution of point centromeres. [ABSTRACT FROM AUTHOR]