Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae

BackgroundAlthough the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown.MethodsWe examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions.ResultsWe demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost.Conclusions and general significanceOur results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.     

Muscle and liver protein synthesis and degradation in growing rats fed a raw field bean (Vicia faba L.) diet

Body weight gain, food intake, gastrocnemius muscle and liver weight, protein and RNA content, as well as the fractional rates of muscle and liver protein synthesis (ks, according to the method of constant infusion of L-[14C]tyrosine), growth (kg) and degradation (kd), along with RNA activity (g of protein synthesized per day/g RNA) of both organs, were determined in growing male rats fed ad libitum over a period of 10 days on 18.7% protein diets containing either casein (5% of methionine added) (control) or the raw legume field bean (Vicia faba L.) as the sole sources of protein. It has been found that as compared to control rats, those fed the raw legume diet exhibited a significant reduction in the rate of growth, muscle RNA, ks, kg, kd and RNA activity, and a significant increase in liver ks, kd and RNA activity. All differences were statistically significant at least at the 5% level. The possible nature of these findings is discussed.     

Control of the Protein Turnover Rates in Lemna minor

The control of protein turnover in Lemna minor has been examined using a method described in the previous paper for determining the rate constants of synthesis and degradation of protein. If Lemna is placed on water, there is a reduction in the rate constants of synthesis of protein and an increase (3- to 6-fold) in the rate constant of degradation. The net effect is a loss of protein from the tissue. Omission of nitrate, phosphate, sulfate, magnesium, or calcium results in increases in the rate constant of degradation of protein.     

Model for Stress-induced Protein Degradation in Lemna minor

Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins.     

The role of mRNA and protein stability in gene expression

How important is the stability of gene products in the process of gene expression? We use a dual-compartment mathematical model to demonstrate the effects that changing the rates of synthesis and degradation of hypothetical mRNAs and proteins would have on the final concentration of protein. The model predicts that the concentration of protein at steady state equals the product of the rate constants for synthesis of mRNA and protein (ks1 and ks2) divided by the product of the rate constants for degradation (kd1 and kd2) and that the rate at which protein concentration changes depends on the rate constants for degradation of both the mRNA and the protein. This permits great flexibility in controlling induction kinetics for particular gene products, since their synthesis, translation, and degradation may be regulated coordinately to permit induction to be stable or transient or to amplify the final yield of protein. We suggest single exons may encode structural features that cause both mRNAs and proteins to be labile, thereby ensuring that modal stabilities of highly regulated macromolecules are similar.     

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.     

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss

Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [³H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [³H]leucine or [¹⁴C]proline.

Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.

Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.

     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

Effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase of rat.

The effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200 mg per 100 g body weight. Catalase activity decreased to about 35% of that of the control at 42-48 h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96 h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degradation (kd) decreased to about 50% of the control.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Replacement perfusion of cultured eucaryotic cells: A method for the accurate measurement of the rates of growth,protein synthesis,and protein turnover

The fractional rates of protein synthesis (k_s) and degradation (k_p) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (k_g). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific-radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship k_g = k_s – k_p. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (k_g = 0.017/hr) turnover represented a major fraction of total synthesis (k_p = 0.032/hr = 0.65ks). At rapid growth rates (k_g = 0.058/hr) the value of k_p was less than 0.005/hr. No major difference was observed between the k_s determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electro-phoresis) from rapid- and slow-growing cultures. Thus, with an invariable k_s, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and WUS totally inhibited by addition of lcupcptin (10 μM) and pepstatin (2 μM), the specific inhibitors of lysosomal cathepsins B (L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appearsto be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH ₄Cl (10 mM) is added to cells growing with a k_g of 0.018/hr ± 0.001 (k_s = 0.050/hr ± 0.002) the growth rate increased to 0.051/hr ± 0.002 without change in the rate of protein synthesis (k_s = 0.049/hr ± 0.003). It is suggested from our data that the cellular growth rate of SP2/0-AG14 is regulated by the lysosomal apparatus; whether this regulation is itself regulated by either a specific compartmentalization of the lysosomal proteinases and/or their substrates or by endogenous protease inhibitors, should prove to be an exciting area for future investigation.     

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.     

The effect of deuterium oxide on protein turnover in Lemna minor

Lemna minor fronds transferred to a sterile culture medium containing 50% (v/v) deuterium oxide (²H₂O) rapidly undergo a loss of soluble protein with a corresponding increase in free amino acids. The loss of protein is due to two factors: (i) the inhibition of protein synthesis for 4 h followed by a slower rate of synthesis than normal, (ii) a rapid 9–10 fold increase in protein degradation. In plants grown for longer periods (3–6 days) in 50% ²H₂O medium, protein synthesis is inhibited by 20% and the rate constant of degradation is 2–3 times that measured in fronds growing in normal (H₂O containing) complete medium. The initial loss of protein is not due to the breakdown of any specific protein fraction. Investigation of several enzymes indicates that all proteins are catabolised in response to ²H₂O treatment. The implications of these results with regard to the interpretation of density-labelling experiments are discussed.     

Macromolecular Synthesis and Degradation in Arthrobacter During Periods of Nutrient Deprivation

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.     

Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg · g⁻¹) compared to the fusion protein SpA-βgalactosidase (138 mg · g⁻¹) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the 1st order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg · g⁻¹ to 120 mg · g⁻¹. The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg · g⁻¹ in 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.     

Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron

Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm.