The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.
During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.Synthesis of mRNA is a multistep process of transcription and pre-mRNA processing. The study of mRNA synthesis has proceeded largely through a reductionist approach, with mRNA production viewed as a series of reactions connected by their substrates and products. It is becoming clear that many RNA-processing reactions occur during transcription elongation (reviewed in references 34 and 45).However, our understanding of the elongation phase of transcription is incomplete. In vitro, two general transcription elongation factors, TFIIF and TFIIS, are sufficient to stimulate in vivo rates of elongation on naked DNA templates (25). In contrast, elongation on nucleosome-bound templates is inefficient, even in the presence of TFIIF and TFIIS, suggesting a requirement for other factors (9,25,26). Several factors have been implicated in the regulation of transcription elongation through chromatin. Among these is the yeast Spt4-Spt5 complex, known as DSIF in human cells (21,57). DSIF/Spt4-Spt5 can inhibit and promote elongation of RNA polymerase II (Pol II) on cellular genes and is required for the stimulation of transcription elongation by human immunodeficiency virus type 1 Tat in vitro (24,28,57,64). A second elongation factor, Spt6, interacts genetically with SPT4, SPT5, and TFIIS and also promotes Tat function in vitro (21, 51, 64). Consistent with their playing a role in elongation, chromatin immunoprecipitation experiments show that the Spt5 and Spt6 proteins associate with transcribed genes in yeast and Drosophila (2, 27, 44). Finally, genetic and biochemical studies of Spt4, Spt5, and Spt6 in yeast have led to the proposal that they function by affecting chromatin structure (6, 51). A third protein complex, FACT, composed of the human Spt16 and SSRP1 proteins, promotes elongation by Pol II through nucleosomes in vitro (40, 41). Its yeast homolog, the CP (or SPN) complex, is composed ...
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.
It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.
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