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Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC’s 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of ‘columns’. These findings provide clues to the evolutionary origins of the NPC. Gatekeeper for the Nucleus The nuclear pore complex plays a crucial role in the cell, as gatekeeper for traffic between the cytoplasm and the interior of the nucleus. It is a large supramolecular complex made up of multiple copies of about 30 different proteins — 456 protein molecules in all. Cell biologists would love to know how each of the pore molecules are placed, but so far this has eluded conventional structural studies. Now, a new proteomics-based technique has provided a detailed view of the architecture of the yeast nuclear pore complex. Half of the complex is made of a core scaffold forming a network coating the surface of the nuclear envelope membrane within which the complex is embedded. The selective transport barrier is formed by the many proteins lining the inner face of the scaffold. Despite its size, there are only a few structural modules in the complex; this underlying simplicity provides possible pointers to an evolutionary origin from a 'primordial' nuclear pore complex. In the cover graphic, the 100-nm diameter pores are shown in the silver-grey nuclear envelope.

The histone H3K36 methylation mark is associated with coding regions of actively transcribed genes, yet it plays a negative role during transcription elongation. In vitro and in vivo studies in budding yeast now reveal that the Isw1b chromatin remodeler is recruited by H3K36 methylation to open reading frames, where it acts in conjunction with a second remodeler to prevent histone exchange and maintain chromatin integrity during transcription elongation. Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, but it has a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro . Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions. In this way, Isw1b and Chd1 are important in maintaining chromatin integrity during transcription elongation by RNAPII.

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies. Gatekeeper for the Nucleus The nuclear pore complex plays a crucial role in the cell, as gatekeeper for traffic between the cytoplasm and the interior of the nucleus. It is a large supramolecular complex made up of multiple copies of about 30 different proteins — 456 protein molecules in all. Cell biologists would love to know how each of the pore molecules are placed, but so far this has eluded conventional structural studies. Now, a new proteomics-based technique has provided a detailed view of the architecture of the yeast nuclear pore complex. Half of the complex is made of a core scaffold forming a network coating the surface of the nuclear envelope membrane within which the complex is embedded. The selective transport barrier is formed by the many proteins lining the inner face of the scaffold. Despite its size, there are only a few structural modules in the complex; this underlying simplicity provides possible pointers to an evolutionary origin from a 'primordial' nuclear pore complex. In the cover graphic, the 100-nm diameter pores are shown in the silver-grey nuclear envelope.

Mass spectrometry is a powerful technology for the analysis of large numbers of endogenous proteins. However, the analytical challenges associated with comprehensive identification and relative quantification of cellular proteomes have so far appeared to be insurmountable. Here, using advances in computational proteomics, instrument performance and sample preparation strategies, we compare protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spans more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins. Stable-isotope labelling by amino acids in cell culture (SILAC) quantification was very accurate across the proteome, as demonstrated by one-to-one ratios of most yeast proteins. Key members of the pheromone pathway were specific to haploid yeast but others were unaltered, suggesting an efficient control mechanism of the mating response. Several retrotransposon-associated proteins were specific to haploid yeast. Gene ontology analysis pinpointed a significant change for cell wall components in agreement with geometrical considerations: diploid cells have twice the volume but not twice the surface area of haploid cells. Transcriptome levels agreed poorly with proteome changes overall. However, after filtering out low confidence microarray measurements, messenger RNA changes and SILAC ratios correlated very well for pheromone pathway components. Systems-wide, precise quantification directly at the protein level opens up new perspectives in post-genomics and systems biology.

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

A survey of 1,590 putative integral, peripheral and lipid-anchored membrane proteins from Saccharomyces cerevisiae reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome. Mapping membrane protein interactions Affinity purification procedures have been successfully used to characterize soluble protein complexes, but complexes involving membrane proteins are more difficult to purify due to their hydrophobic nature. Here, Andrew Emili and colleagues have affinity purified membrane proteins from the yeast Saccharomyces cerevisiae in the presence of three different non-denaturing detergents, and identified the co-purifying proteins by mass spectrometry. They generate an extensive physical interaction map of membrane protein interactions, most of which have not been previously reported. Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases 1 . Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae , which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein–protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

High-throughput peptide synthesis and mass spectrometry are used to generate a near-complete reference map of the Saccharomyces cerevisiae proteome; two versions of the map (supporting discovery- and hypothesis-driven proteomics) are then applied to a protein-based quantitative trait locus analysis. A global map of yeast proteins Complete 'gold standard' reference maps of the components within a system are valuable resources for a research community. This paper presents one such resource, a complete mass-spectrometric reference map of the budding yeast Saccharomyces cerevisiae . The map comes in two versions — one for discovery-driven (shotgun) and the other for hypothesis-driven (targeted) proteomic measurements — and will support most studies performed with contemporary proteomic technologies. The maps provide essentially a set of highly specific assays for the detection and quantification of every yeast protein in any sample, and their value is demonstrated here in a protein quantitative trait locus analysis. Experience from different fields of life sciences suggests that accessible, complete reference maps of the components of the system under study are highly beneficial research tools. Examples of such maps include libraries of the spectroscopic properties of molecules, or databases of drug structures in analytical or forensic chemistry. Such maps, and methods to navigate them, constitute reliable assays to probe any sample for the presence and amount of molecules contained in the map. So far, attempts to generate such maps for any proteome have failed to reach complete proteome coverage 1 , 2 , 3 . Here we use a strategy based on high-throughput peptide synthesis and mass spectrometry to generate an almost complete reference map (97% of the genome-predicted proteins) of the Saccharomyces cerevisiae proteome. We generated two versions of this mass-spectrometric map, one supporting discovery-driven (shotgun) 3 , 4 and the other supporting hypothesis-driven (targeted) 5 , 6 proteomic measurements. Together, the two versions of the map constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. To show the utility of the maps, we applied them to a protein quantitative trait locus (QTL) analysis 7 , which requires precise measurement of the same set of peptides over a large number of samples. Protein measurements over 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, influencing the levels of related proteins. Our results suggest that selective pressure favours the acquisition of sets of polymorphisms that adapt protein levels but also maintain the stoichiometry of functionally related pathway members.

A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein–protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

It remains unclear how proteins translocate across the peroxisomal membrane. Insights into a potential import pore are provided with the finding that the import receptor Pex5p forms a dynamic ion channel together with Pex14p, which can be induced to open upon receptor-cargo complex association. The peroxisomal protein import machinery differs fundamentally from known translocons (endoplasmic reticulum, mitochondria, chloroplasts, bacteria) as it allows membrane passage of folded, even oligomerized proteins 1 . However, the mechanistic principles of protein translocation across the peroxisomal membrane remain unknown. There are various models that consider membrane invagination events, vesicle fusion or the existence of large import pores. Current data show that a proteinaceous peroxisomal importomer enables docking of the cytosolic cargo-loaded receptors, cargo translocation and receptor recycling 2 . Remarkably, the cycling import receptor Pex5p changes its topology from a soluble cytosolic form to an integral membrane-bound form. According to the transient pore hypothesis, the membrane-bound receptor is proposed to form the core component of the peroxisomal import pore 3 . Here, we demonstrate that the membrane-associated import receptor Pex5p together with its docking partner Pex14p forms a gated ion-conducting channel which can be opened to a diameter of about 9 nm by the cytosolic receptor–cargo complex. The newly identified pore shows striking dynamics, as expected for an import machinery translocating proteins of variable sizes.

To increase the range and precision of genetic interaction studies in Saccharomyces cerevisiae , a collection of hypomorphic alleles of essential yeast genes and a highly sensitive flow cytometry–based growth competition assay are presented. Also in this issue, Yan et al . present a similar strain collection, tagged with unique bar-code identifiers, and use this collection in pooled chemical genetic screens. Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.