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The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ~30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution. [ABSTRACT FROM AUTHOR]

Despite the appreciable success of synthetic nanomaterials for targeted cancer therapy in preclinical studies, technical challenges involving their large-scale, cost-effective production and intrinsic toxicity associated with the materials, as well as their inability to penetrate tumor tissues deeply, limit their clinical translation. Here, we describe biologically derived nanocarriers developed from a bioengineered yeast strain that may overcome such impediments. The budding yeast Saccharomyces cerevisiae was genetically engineered to produce nanosized vacuoles displaying human epidermal growth factor receptor 2 (HER2)-specific affibody for active targeting. These nanosized vacuoles efficiently loaded the anticancer drug doxorubicin (Dox) and were effectively endocytosed by cultured cancer cells. Their cancer-targeting ability, along with their unique endomembrane compositions, significantly enhanced drug penetration in multicellular cultures and improved drug distribution in a tumor xenograft. Furthermore, Dox-loaded vacuoles successfully prevented tumor growth without eliciting any prolonged immune responses. The current study provides a platform technology for generating cancer-specific, tissue-penetrating, safe, and scalable biological nanoparticles for targeted cancer therapy. [ABSTRACT FROM AUTHOR]

Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5' to 3' resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor. [ABSTRACT FROM AUTHOR]

The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription. [ABSTRACT FROM AUTHOR]

The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits -mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)- and two soluble regulatory components- MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery. [ABSTRACT FROM AUTHOR]

The molecular structure of the yeast nuclear pore complex (NPC) and the translocation of model particles have been studied with a molecular theory that accounts for the geometry of the pore and the sequence and anchoring position of the unfolded domains of the nucleoporin proteins (the FG-Nups), which control selective transport through the pore. The theory explicitly models the electrostatic, hydrophobic, steric. conformational. and acid-base properties of the FG-Nups. The electrostatic potential within the pore, which arises from the specific charge distribution of the FG-Nups, is predicted to be negative close to pore walls and positive along the pore axis. The positive electrostatic potential facilitates the translocation of negatively charged particles, and the free energy barrier for translocation decreases for increasing particle hydro-phobicity. These results agree with the experimental observation that transport receptors that form complexes with hydrophilic/neutral or positively charged proteins to transport them through the NPC are both hydrophobic and strongly negatively charged. The molecular theory shows that the effects of electrostatic and hydrophobic interactions on the translocating potential are cooperative and nonequivalent due to the interaction-dependent reorganization of the FG-Nups in the presence of the translocating particle. The combination of electrostatic and hydrophobic interactions can give rise to complex translocation potentials displaying a combination of wells and barriers, in contrast to the simple barrier potential observed for a hydrophilic/neutral translocating particle. This work demonstrates the importance of explicitly considering the amino acid sequence and hydrophobic, electrostatic, and steric interactions in understanding the translocation through the NPC. [ABSTRACT FROM AUTHOR]

The accuracy of the 3'-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of hetero-dimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3'-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis. [ABSTRACT FROM AUTHOR]

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research. [ABSTRACT FROM AUTHOR]

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a single-sequenced sample. [ABSTRACT FROM AUTHOR]

The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dami complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Daml also forms smaller, nonring patches in vitro that track the dynamic ends of MTs. However, the identity of Dam1's functional form in vivo remains unknown. Here we report a comprehensive in vivo characterization of Daml in the fission yeast Schizosaccharomyces pombe. In addition to their dense localizations on kinetochores and spindle MTs during mitosis, we identify that Daml is also localized onto cytoplasmic MTs as discrete spots in interphase, providing the unique opportunity to analyze Dami oligomers at the single-particle resolution in live cells. Such analysis shows that each oligomer contains one to five copies of Dam1, and is able to "switch-rail" while moving along MTs, precluding the possibility of a 16-unit encircling structure., Dam1 patches track the plus ends of the shortening, but not the elongating, MTs and retard MT depolymerization. Together with Ma13, the EB1-like MT-interacting protein, cytoplasmic Dam1 plays an important role in maintaining proper cell shape. In mitosis, kinetochore-associated Dam1 appears to facilitate kMT depolymerization. Together, our findings suggest that patches, instead of rings, are the physiologically functional forms of Dam1 in pombe. Our findings help establish the benchmark parameters of the Dam1 coupler and elucidate the mechanism of its functions. [ABSTRACT FROM AUTHOR]

Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regulating sphingolipid synthesis. Phospholipid homeostasis is also affected in orm1Δ orm2Δ cells: the cells are inositol auxotrophs with impaired transcriptional regulation of genes encoding phospholipid biosynthesis enzymes. Strikingly, impaired growth of orm1Δ orm2Δ cells is associated with constitutive unfolded protein response, sensitivity to stress, and slow ER- to-Golgi transport. Inhibition of sphingolipid synthesis suppresses orm1Δ orm2Δ phenotypes, including ER stress, suggesting that disrupted sphingolipid homeostasis accounts for pleiotropic phenotypes. Thus, the yeast Orm proteins control membrane biogenesis by coordinating lipid homeostasis with protein quality control. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved Smc5/6 complex is implicated in recombinational repair, but its function in this process has been elusive. Here we report that the budding yeast SmcS/6 complex directly binds to the DNA helicase Mph1. Mph1 and its helicase activity define a replication-associated recombination subpathway. We show that this is toxic when the Smc5/6 complex is defective, because mph1Δ and its helicase mutations suppress multiple defects in mutants of the Smc5/6 complex, including their sensitivity to replication-blocking agents, growth defects, and inefficient chromatid separation, whereas MPH1 overexpression exacerbates some of these defects. We further demonstrate that Mph1 and its helicase activity are largely responsible for the accumulation of potentially deleterious recombination intermediates in mutants of the Smc5/6 complex. We also present evidence that mph1Δ does not alleviate sensitivity to DNA damage or the accumulation of recombination intermediates in cells lacking Sgs1, which is thought to function together with the Smc5/6 complex. Thus, our results reveal a function of the SmcS/6 complex in the Mph1-dependent recombinational subpathway that is distinct from Sgs1. We suggest that the Smc5/6 complex can counteract/ modulate a pro-recombinogenic function of Mph1 or facilitate the resolution of recombination structures generated by Mph1. [ABSTRACT FROM AUTHOR]

We show that, during budding yeast meiosis, axis ensemble Hopi/Redi and synaptonemal complex (SC) component Zipi tend to occur in alternating strongly staining domains. The widely conserved AAA+-ATPase Pch2 mediates this pattern, likely by means of direct intervention along axes. Pch2 also coordinately promotes timely progression of cross-over (CO) and noncross-over (NCO) recombination. Oppositely, in a checkpoint-triggering aberrant situation (zip1Ɗ), Pch2 mediates robust arrest of stalled recombination complexes, likely via nucleolar localization. We suggest that, during WT meiosis, Pch2 promotes progression of SC-associated CO and NCO recombination complexes at a regulated early-midpachytene transition that is rate-limiting for later events; in contrast, during defective meiosis, Pch2 ensures that aberrant recombination complexes fail to progress so that intermediates can be harmlessly repaired during eventual return to growth. Positive vs. negative roles of Pch2 in the two situations are analogous to positive vs. negative roles of Mecl/ATR, suggesting that Pch2 might mediate Mecl/ ATR activity. We further propose that regulatory surveillance of normal and abnormal interchromosomal interactions in mitotic and meiotic cells may involve "structure-dependent interchromosomal interaction" (SDIX) checkpoints. [ABSTRACT FROM AUTHOR]

Presents a study that used two-hybrid screening to deduce a protein-protein interaction map of the yeast RNA polymerase III of Saccharomyces cerevisiae. Methodology of the study; Results and discussion; Conclusion.

Presents an analysis marginal fitness contributions of nonessential genes in yeast. Discussion on the complete study on Saccharomyces cerevisiae; Information on the significant contributions being made to fitness under routine growth conditions; Examination of 34 strains with disruptions in nonessential genes.

The author discusses the article by S. C. Weber and colleagues on active fluctuating motion in bacteria and yeast's cellular nucleus. He says that the authors show that such motion is nonthermal, but is proportional to temperature and differs inversely with viscoelastic resistance to the medium. He says that it suggests that diffusion shows the balance between thermal driving force and viscous resistance. He says that the research may serve as avenue of research to define cytoskeletal activity.

The article focuses on kinetic analysis of ribosome recycling and translation termination coupling in yeast. It says that the process consists of discrete steps including elongation, initiation, and recycling, in which recycling is needed for ribosome complex disassembly in protein synthesis. It states the use of biochemical approaches to define the processes and suggest mechanistic framework. Results show the kinetic system of translation and recycling events integration in eukaryotes.

The article presents a study which investigates the correlation of nonrespiratory mitochondrial function and oxidative stress response in starvation survival of yeast. The study evaluated the metabolite abundance, gene expression, and the phenotype of the yeast during metabolite starvation. The study shows that the yeast's survival correlates with its ability to fight oxidative stress and its oxidative metabolism might increase the antioxidants' abundance that could benefit the cells.

An interview with Rosenstiel Basic Medical Sciences Research Center director James E. Haber is presented. Haber states that Saccharomyces cereviciae is a unicellular organism which attracts the interests of people who are studying DNA repair. He mentions the mating type switching (MAT) in yeast chromosomes which specifies the mating types of haploid cells that are analogous for male and female. Moreover, he says that their work could help ensure the ability of cells to repair DNA.

A correction to the article "Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space," by Shin Kawano, Koji Yamano, Mari Naoé, Takaki Momose, Kayoko Terao, Shuh-ichi Nishikawa, Nobuhisa Watanabe and Tishiya Endo that was published in the August 10, 2009 issue of Proc Natl Acad Sci USA is presented.