Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach

Evidence for complete and partial surface renewal at an air-water interface

A wind-wave flume is used to determine the extent to which the thermal boundary layer (TBL) at a wind-forced air-water interface is completely renewed from below. We measure skin temperature, Tskin, radiometrically, temperature immediately below the TBL, Tsubskin, using a temperature profiler, and net heat flux using the gradient flux technique. The Tskin probability density function, p(Tskin), and surface renewal time scale, τ, were measured using passive and active infrared imaging techniques, respectively. We find that the mean percentile rank of Tsubskin in p(Tskin) is 99.90, implying that complete surface renewal occurs. This result suggests an alternative to radiometric measurement of Tskin through the simple combination of an infrared camera and an in situ temperature sensor. Comparison of the temperature difference across the TBL to the expected cooling implies that a significant portion of events only partially renew the TBL. This result should impact efforts to improve air-sea transfer models

Statistics of surface divergence and their relation to air-water gas transfer velocity

Air-sea gas fluxes are generally defined in terms of the air/water concentration difference of the gas and the gas transfer velocity,kL. Because it is difficult to measure kLin the ocean, it is often parameterized using more easily measured physical properties. Surface divergence theory suggests that infrared (IR) images of the water surface, which contain information concerning the movement of water very near the air-water interface, might be used to estimatekL. Therefore, a series of experiments testing whether IR imagery could provide a convenient means for estimating the surface divergence applicable to air-sea exchange were conducted in a synthetic jet array tank embedded in a wind tunnel. Gas transfer velocities were measured as a function of wind stress and mechanically generated turbulence; laser-induced fluorescence was used to measure the concentration of carbon dioxide in the top 300 μm of the water surface; IR imagery was used to measure the spatial and temporal distribution of the aqueous skin temperature; and particle image velocimetry was used to measure turbulence at a depth of 1 cm below the air-water interface. It is shown that an estimate of the surface divergence for both wind-shear driven turbulence and mechanically generated turbulence can be derived from the surface skin temperature. The estimates derived from the IR images are compared to velocity field divergences measured by the PIV and to independent estimates of the divergence made using the laser-induced fluorescence data. Divergence is shown to scale withkLvalues measured using gaseous tracers as predicted by conceptual models for both wind-driven and mechanically generated turbulence

Формування законодавчої і нормативної бази архівної справи в республіці Казахстан

Аналізується законодавство Республіки Казахстан, спрямоване на забезпечення збереження документальної спадщини, регулювання діяльності архівної системи в умовах державного суверенітету. Визначаються особливості архівного законодавства кінця 1990-х рр., прослідковуються його зміни та напрями удосконалення в 2000-х рр., процеси формування нормативної бази архівної справи.Анализируется законодательство Республики Казахстан, направленное на обеспечение сохранности документального наследия, регулирование деятельности архивной системы в условиях государственного суверенитета. Определяются особенности архивного законодательства конца 1990-х гг., прослеживаются его изменения и направления усовершенствования в 2000- х гг., процессы формирования нормативной базы архивного дела.The legislation of Republic of Kazakhstan, directed on providing of safety of documentary legacy, adjusting of activity of the archival system in the conditions of the state sovereignty is analysed in the article. The features of the archival legislation the end of 1990-th are determined, its changes and directions of improvement in the 2000-th, the processes of forming of normative base of the archival affairs are traced

Advanced EFL learners' beliefs about language learning and teaching: a comparison between grammar, pronunciation, and vocabulary

This paper reports on the results of a study exploring learners’ beliefs on the learning and teaching of English grammar, pronunciation and vocabulary at tertiary level. While the importance of learners’ beliefs on the acquisition process is generally recognized, few studies have focussed on and compared learners’ views on different components of the language system. A questionnaire containing semantic scale and Likert scale items probing learners’ views on grammar, pronunciation and vocabulary was designed and completed by 117 native speakers of Dutch in Flanders, who were studying English at university. The analysis of the responses revealed that (i) vocabulary was considered to be different from grammar and pronunciation, both in the extent to which an incorrect use could lead to communication breakdown and with respect to the learners’ language learning strategies, (ii) learners believed in the feasibility of achieving a native-like proficiency in all three components, and (iii) in-class grammar, pronunciation and vocabulary exercises were considered to be useful, even at tertiary level. The results are discussed in light of pedagogical approaches to language teaching

Mechanisms of PTPσ-Mediated Presynaptic Differentiation

Formation of synapses between neurons depends in part on binding between axonal and dendritic cell surface synaptic organizing proteins, which recruit components of the developing presynaptic and postsynaptic specializations. One of these presynaptic organizing molecules is protein tyrosine phosphatase σ (PTPσ). Although the protein domains involved in adhesion between PTPσ and its postsynaptic binding partners are known, the mechanisms by which it signals into the presynaptic neuron to recruit synaptic vesicles and other necessary components for regulated transmitter release are not well understood. One attractive candidate to mediate this function is liprin-α, a scaffolding protein with well-established roles at the synapse. We systematically mutated residues of the PTPσ intracellular region (ICR) and used the yeast dihydrofolate reductase (DHFR) protein complementation assay to screen for disrupted interactions between these mutant forms of PTPσ and its various binding partners. Using a molecular replacement strategy, we show that disrupting the interaction between PTPσ and liprin-α, but not between PTPσ and itself or another binding partner, caskin, abolishes presynaptic differentiation. Furthermore, phosphatase activity of PTPσ and binding to extracellular heparan sulfate (HS) proteoglycans are dispensable for presynaptic induction. Previous reports have suggested that binding between PTPσ and liprin-α is mediated by the PTPσ membrane-distal phosphatase-like domain. However, we provide evidence here that both of the PTPσ phosphatase-like domains mediate binding to liprin-α and are required for PTPσ-mediated presynaptic differentiation. These findings further our understanding of the mechanistic basis by which PTPσ acts as a presynaptic organizer

Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R2 = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration

Physiological Correlates of Volunteering

We review research on physiological correlates of volunteering, a neglected but promising research field. Some of these correlates seem to be causal factors influencing volunteering. Volunteers tend to have better physical health, both self-reported and expert-assessed, better mental health, and perform better on cognitive tasks. Research thus far has rarely examined neurological, neurochemical, hormonal, and genetic correlates of volunteering to any significant extent, especially controlling for other factors as potential confounds. Evolutionary theory and behavioral genetic research suggest the importance of such physiological factors in humans. Basically, many aspects of social relationships and social activities have effects on health (e.g., Newman and Roberts 2013; Uchino 2004), as the widely used biopsychosocial (BPS) model suggests (Institute of Medicine 2001). Studies of formal volunteering (FV), charitable giving, and altruistic behavior suggest that physiological characteristics are related to volunteering, including specific genes (such as oxytocin receptor [OXTR] genes, Arginine vasopressin receptor [AVPR] genes, dopamine D4 receptor [DRD4] genes, and 5-HTTLPR). We recommend that future research on physiological factors be extended to non-Western populations, focusing specifically on volunteering, and differentiating between different forms and types of volunteering and civic participation

An Assembly of Proteins and Lipid Domains Regulates Transport of Phosphatidylserine to Phosphatidylserine Decarboxylase 2 in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

Background: The machinery for interorganelle phosphatidylserine (PtdSer) transport is poorly defined at the molecular level. Results: Molecular interaction studies identify specific protein-protein and protein-lipid interactions in PtdSer transport. Conclusion: A protein and lipid interaction network defines key participants in PS transport to the locus of PtdSer-decarboxylase 2. Significance: This study identifies important molecular participants involved in non-vesicular phospholipid traffic