The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.During and after eukaryotic transcription, RNA is packaged into RNP complexes, then modified, spliced, folded and exported into the cytoplasm through nuclear pore complexes (NPCs). There are two main forms of RNPs. Each contains a different type of RNA: mRNPs contain mRNA, and rRNPs contain ribosomal RNA (rRNA). Each also uses discrete processing factors, which associate with RNPs in a dynamic fashion to define the order of transcript maturation. The function and composition of many of these RNP complexes, however, is poorly understood.In recent years, there have been considerable efforts to study the composition of mRNPs and rRNPs in greater detail. A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export. Here we describe a method that aims to overcome these limitations and capture the dynamic interactome of maturing RNP complexes.We used this method to analyze complexes along both the ribosome and mRNA biogenesis pathways. RESULTSA strategy for isolation of RNP complexes Our strategy comprises seven steps that we optimized for efficient isolation of intact RNP complexes ( Fig. 1 and Supplementary Methods online). Collected cells are rapidly frozen in liquid nitrogen and broken open in the solid phase by milling 5 , using a planetary ball mill that produces particles of B1-2 mm in diameter (small enough for rapid and efficient RNP extraction). Because cell breakage occurs in the frozen state, we avoid damage to RNP complexes by released nucleases and proteases, as well as redistribution of proteins within the extract. The resulting frozen grindate is rapidly thawed into an RNP-compatible buffer (see Supplementary Methods), to produce an extract that is then immediately clarified by filtration. We isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose. The magnetic beads are small (2.8 mm) and so have a large surface area-to-volume ratio that increases the speed of binding. Moreover, the beads are impermeable, such that the antibodies are densely conjugated to their exposed surface; hence, there is no theoretical upper limit to the size of isolated complexes (unlike with permeable resins such as Sepharose, which are limited by their pore size). Incubation times...

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

SummaryPost-translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a 'histone code' that specifies epigenetic information for transcription regulation. With a limited number of histone-modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone-modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0-specific masking of the site, owing to non-covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.

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The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro , with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT SEB1 , successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

No abstract

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1, 2 . The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2 . Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3 . It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement. ProtocolIt is very important to wear powder-free gloves during the preparation of the thin layer.