Protein activity is the ultimate arbiter of function in most cellular pathways, and protein concentration is fundamentally connected to protein action. While the proteome of yeast has been subjected ...to the most comprehensive analysis of any eukaryote, existing datasets are difficult to compare, and there is no consensus abundance value for each protein. We evaluated 21 quantitative analyses of the S. cerevisiae proteome, normalizing and converting all measurements of protein abundance into the intuitive measurement of absolute molecules per cell. We estimate the cellular abundance of 92% of the proteins in the yeast proteome and assess the variation in each abundance measurement. Using our protein abundance dataset, we find that a global response to diverse environmental stresses is not detected at the level of protein abundance, we find that protein tags have only a modest effect on protein abundance, and we identify proteins that are differentially regulated at the mRNA abundance, mRNA translation, and protein abundance levels.
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•Meta-analysis defines the protein abundance distribution of the yeast proteome•Low- and high-abundance proteins are enriched for biological functions•Stress-dependent abundance changes reveal functional connections•Protein fusion tags have a limited effect on native protein abundance
By normalizing and converting 21 protein abundance datasets to the intuitive unit of molecules per cell, we provide precise and accurate abundance estimates for 92% of the yeast proteome. Our protein abundance dataset proves useful for exploring the cellular response to environmental stress, the balance between transcription and translation in regulating protein abundance, and the systematic evaluation of the effect of protein tags on protein abundance.
Fusarium wilt, caused by the fungus
f.sp.
(
), is one of the most important and destructive diseases in banana crops worldwide. There have been numerous studies into the infection process of this ...soil-borne pathogen; however, the extent of research into the movement of the pathogen through the rhizome and into the rest of the plant is limited. Furthermore, little is known about the movement of the pathogen once it reaches the aerial components of the plant. A strain of
sub-tropical race 4, genetically transformed with green fluorescent protein (GFP) gene, was used to monitor the movement of the pathogen through two susceptible cultivars, Cavendish 'Williams' (
AAA) and Lady Finger (
AAB). Visualization of the pathogen
demonstrated its presence in the roots, the rhizome and in the outer leaf sheaths of the pseudostem prior to the appearance of external symptoms. Within the non-senescing leaf sheaths, the migration of
was confined to the xylem vessels; this included those where the vascular tissue was visibly discolored, as well as those, which were apparently healthy. As senescence of leaf sheaths occurred, chlamydospores developed within the gas spaces, while formation of sporodochia, and hyphal growth were apparent on the outer surface of senescing leaf sheaths. These results generate a greater understanding of the epidemiology of
, providing much needed knowledge to assist in the future management of Fusarium wilt incursions, as well as enhancing protocols for ongoing on-farm hygiene and biosecurity.
Accumulating evidence has linked pathological changes associated with chronic alcohol exposure to neuroimmune signaling mediated by microglia. Prior characterization of the microglial ...structure–function relationship demonstrates that alterations in activity states occur concomitantly with reorganization of cellular architecture. Accordingly, gaining a better understanding of microglial morphological changes associated with ethanol exposure will provide valuable insight into how neuroimmune signaling may contribute to ethanol‐induced reshaping of neuronal function. Here we have used Iba1‐staining combined with high‐resolution confocal imaging and 3D reconstruction to examine microglial structure in the prelimbic (PL) cortex and nucleus accumbens (NAc) in male Long‐Evans rats. Rats were either sacrificed at peak withdrawal following 15 days of exposure to chronic intermittent ethanol (CIE) or 24 hr after two consecutive injections of the immune activator lipopolysaccharide (LPS), each separated by 24 hr. LPS exposure resulted in dramatic structural reorganization of microglia in the PL cortex, including increased soma volume, overall cellular volume, and branching complexity. In comparison, CIE exposure was associated with a subtle increase in somatic volume and differential effects on microglia processes, which were largely absent in the NAc. These data reveal that microglial activation following a neuroimmune challenge with LPS or exposure to chronic alcohol exhibits distinct morphometric profiles and brain region‐dependent specificity.
Microglia structural reorganization, and altered neuroimmune signaling, are implicated in pathological adaptations that promote excessive drinking. Here we used Iba1 immunohistochemistry, high‐resolution confocal microscopy, and digital reconstruction analyses of Iba1‐positive microglia in the prelimbic (PL) cortex and nucleus accumbens (NAc) of rats during withdrawal from chronic intermittent ethanol (CIE) exposure, rats injected with lipopolysaccharide (LPS), and an ethanol‐naïve (air)‐exposed group. We found that CIE and LPS led to dichotomous alterations in microglia structural complexity in the PL cortex that were largely absent in the NAc. These findings add to a growing body of literature pointing towards altered microglia structural complexity, and presumably function, in ethanol‐induced adaptations in the brain.
Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists ...using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.
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•Method to follow protein unfolding in real-time with atomic resolution.•Unfolding intermediates are not present in solution at detectable levels.•Protein degradation is tightly ...coupled to unfolding.
Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN–20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution.
Plasmolysis: Loss of Turgor and Beyond Lang, Ingeborg; Sassmann, Stefan; Schmidt, Brigitte ...
Plants (Basel),
11/2014, Letnik:
3, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Plasmolysis is a typical response of plant cells exposed to hyperosmotic stress. The loss of turgor causes the violent detachment of the living protoplast from the cell wall. The plasmolytic process ...is mainly driven by the vacuole. Plasmolysis is reversible (deplasmolysis) and characteristic to living plant cells. Obviously, dramatic structural changes are required to fulfill a plasmolytic cycle. In the present paper, the fate of cortical microtubules and actin microfilaments is documented throughout a plasmolytic cycle in living cells of green fluorescent protein (GFP) tagged Arabidopsis lines. While the microtubules became wavy and highly bundled during plasmolysis, cortical filamentous actin remained in close vicinity to the plasma membrane lining the sites of concave plasmolysis and adjusting readily to the diminished size of the protoplast. During deplasmolysis, cortical microtubule re-organization progressed slowly and required up to 24 h to complete the restoration of the original pre-plasmolytic pattern. Actin microfilaments, again, recovered faster and organelle movement remained intact throughout the whole process. In summary, the hydrostatic skeleton resulting from the osmotic state of the plant vacuole "overrules" the stabilization by cortical cytoskeletal elements.
In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN ...FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves.
Analysis of synthesis mutants demonstrates an ascorbate requirement for growth under low light and for high light-dependent anthocyanin accumulation, but no consistent effects on photoinhibition or ...zeaxanthin accumulation were found.
Abstract
The requirements for ascorbate for growth and photosynthesis were assessed under low (LL; 250 µmol m-2 s-1) or high (HL; 1600 µmol m-2 s-1) irradiance in wild-type Arabidopsis thaliana and two ascorbate synthesis mutants (vtc2-1 and vtc2-4) that have 30% wild-type ascorbate levels. The low ascorbate mutants had the same numbers of leaves but lower rosette area and biomass than the wild type under LL. Wild-type plants experiencing HL had higher leaf ascorbate, anthocyanin, and xanthophyll pigments than under LL. In contrast, leaf ascorbate levels were not increased under HL in the mutant lines. While the degree of oxidation measured using an in vivo redox reporter in the nuclei and cytosol of the leaf epidermal and stomatal cells was similar under both irradiances in all lines, anthocyanin levels were significantly lower in the low ascorbate mutants than in the wild type under HL. Differences in the photosynthetic responses of vtc2-1 and vtc2-4 mutants were observed. Unlike vtc2-1, the vtc2-4 mutants had wild-type zeaxanthin contents. While both low ascorbate mutants had lower levels of non-photochemical quenching of chlorophyll a fluorescence (NPQ) than the wild type under HL, qPd values were greater only in vtc2-1 leaves. Ascorbate is therefore essential for growth but not for photoprotection.
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•An ITO (Indium Titanium oxide) based GFP (Green Fluorescent Protein) immobilized electrode (GFP/ITO) was developed to study GFP photobleaching electrochemically.•Electrochemical ...Impedance Spectroscopy (EIS) was used to study changes in impedance of immobilized GFP and correlated with decrease in its fluorescence intensity.•Similar studies were performed with Streptavidin-TMR (tetramethylrhodamine) and Bovine Serum Albumin (BSA) immobilized to ITO for comparative analysis.•It is one of the first kind of study to detect fluorescence changes in GFP using an electrochemical approach.
Electron transport and transition within a molecule are involved in some of the major cellular and biochemical processes in an organism. In proteins, electron transfer is generally characterized by two mechanisms, electron tunnelling and electron hopping. Green Fluorescent Protein (GFP) is the most widely used fluorescent protein in various biological studies. We know that folding occurs in GFP followed by an autocatalytic cyclization and finally oxidation to form a chromophore. The fluorescence phenomenon would cease in proteins which have lost their native structure. In this paper, we have immobilized Strep-TMR (Streptavidin-TMR), BSA (Bovine Serum Albumin) and GFP on a solid surface and checked its functionality under a fluorescence microscope. We also measured the impedance of these bound proteins and found out that ωmax of the Nyquist plot in GFP was less than that of Strep-TMR and BSA. However, when GFP was photobleached by light exposure at various time intervals, an increase in ωmax was observed. This photobleaching and impedimetric correlation observed in case of GFP was compared with the other two proteins as well. Studies have suggested that a number of mechanisms are involved in photobleaching, like structural changes due to photodegradation, ceasing of electron transport or the completion of absorption emission cycles of a fluorophore. In our study, it seems that the electrons responsible for fluorescence in GFP might be responsible for the changes in the ωmax of the Nyquist plot of EIS system. This study is one of the first few approaches that has tried to establish a relationship between fluorescence and electrochemical changes within a fluorescent protein upon photoexposure.
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