"It'll never end, I'll never go": Representation of Caregiving in Samuel Beckett's Endgame and Footfalls.

Research on the unrepresentability of death in Samuel Beckett's oeuvre abound in Beckett scholarship, but little attention has been given to the artist's representation of caregiving to the dying in his plays. With reference to Martin Heidegger's concept of care and Albert Camus's idea of the absurd, this article analyzes Endgame (1957) and Footfalls (1976) by attending to Beckett's dramatic representation of caregiving as undergirded by a sense of its absurdity. The almost 20-year gap between the writing of both plays highlights the development of an understanding that this sense of absurdity is never about the caregiver's questioning of one's obligation to the dependent but about how one chooses to respond to caregiving as an absurd predicament. The pertinence of such a representation of caregiving by Beckett lies in its poignant articulation of a complex experience that is often left unexpressed by caregivers who prioritize their dependent loved ones over themselves.

The Critical Role of Equilibrative Nucleoside Transporter-2 in Modulating Cerebral Damage and Vascular Dysfunction in Mice with Brain Ischemia-Reperfusion.

BACKGROUND: Cerebral vascular protection is critical for stroke treatment. Adenosine modulates vascular flow and exhibits neuroprotective effects, in which brain extracellular concentration of adenosine is dramatically increased during ischemic events and ischemia-reperfusion. Since the equilibrative nucleoside transporter-2 (Ent2) is important in regulating brain adenosine homeostasis, the present study aimed to investigate the role of Ent2 in mice with cerebral ischemia-reperfusion. METHODS: Cerebral ischemia-reperfusion injury was examined in mice with transient middle cerebral artery occlusion (tMCAO) for 90 minutes, followed by 24-hour reperfusion. Infarct volume, brain edema, neuroinflammation, microvascular structure, regional cerebral blood flow (rCBF), cerebral metabolic rate of oxygen (CMRO2), and the production of reactive oxygen species (ROS) were examined following the reperfusion. RESULTS: Ent2 deletion reduced the infarct volume, brain edema, and neuroinflammation in mice with cerebral ischemia-reperfusion. tMCAO-induced disruption of brain microvessels was ameliorated in Ent2-/- mice, with a reduced expression of matrix metalloproteinases-9 and aquaporin-4 proteins. Following the reperfusion, the rCBF of the wild-type (WT) mice was quickly restored to the baseline, whereas, in Ent2-/- mice, rCBF was slowly recovered initially, but was then higher than that in the WT mice at the later phase of reperfusion. The improved CMRO2 and reduced ROS level support the beneficial effects caused by the changes in the rCBF of Ent2-/- mice. Further studies showed that the protective effects of Ent2 deletion in mice with tMCAO involve adenosine receptor A2AR. CONCLUSIONS: Ent2 plays a critical role in modulating cerebral collateral circulation and ameliorating pathological events of brain ischemia and reperfusion injury.

Predictors of Response to Oral Medications and Low-Histamine Diet in Patients with Chronic Urticaria.

BACKGROUND: Chronic urticaria (CU) is comprised of diverse phenotypes, and thus, a shift towards a precision medical approach is warranted in its management. METHODS: This study enrolled 78 patients with CU. Serum erythrocyte sedimentation rate, hemoglobin, hematocrit, eosinophil count, IgE, antinuclear antibody (ANA), and serum diamine oxidase (DAO) levels of the patients were measured and were compared according to the patient's response to second-generation antihistamines (sgAH), corticosteroids, leukotriene receptor antagonist (LTRA), H2 blockers, and low-histamine diet. RESULTS: Age- and sex-adjusted logistic regression analysis showed that patients with duration of CU > 3 years (adjusted odd ratio [aOR] = 4.39) and a DAO level < 10 U/mL (aOR = 3.90) were significantly associated with a good sgAH response. Age > 50 years (aOR = 0.02), duration of chronic urticaria > 3 years (aOR =0.06), and an ANA titer ≥ 1 : 80 (aOR = 0.03) were significantly and inversely associated with corticosteroid response. A low-histamine diet response was significantly associated with LTRA response (aOR = 67.29). In addition, a DAO level < 5.4 U/mL (aOR = 71.95) was significantly associated with H2 blocker response. Furthermore, concomitant angioedema (aOR = 10.56), multiple food triggers (aOR = 11.69), and a DAO level < 5.4 U/mL (aOR = 3.78) were significantly associated with a low-histamine diet response. Conversely, dermatographic urticaria and a hematocrit level < 36% were significantly and inversely associated with low-histamine diet response. CONCLUSIONS: Several promising biomarkers were identified in this study to predict the efficacy of chronic urticaria treatment. DAO could be a novel biomarker for predicting the efficacy not only of dietary intervention but also for antagonists of H1 and H2 receptors.

Association between clinical phenotypes of dermatomyositis and polymyositis with myositis-specific antibodies and overlap systemic autoimmune diseases.

ABSTRACT: The aim of this study was to evaluate the association between clinical phenotypes of dermatomyositis (DM) and polymyositis (PM) with myositis-specific antibodies (MSAs), and overlap diagnosis of systemic autoimmune diseases.This cross-sectional study was conducted on 67 patients with DM and 27 patients with PM recruited from a regional hospital in southern Taiwan. Clinical phenotypes of DM and PM were assessed and MSAs were measured using a commercial line blot assay. The association of clinical phenotypes of DM and PM with MSAs and overlap diagnosis of systemic autoimmune diseases was performed using univariate and multiple logistic regression analyses.Clinically, patients with DM and PM and overlap diagnosis of systemic sclerosis were associated with a higher risk of interstitial lung diseases (ILDs) (odds ratio [OR] = 6.73; P = .048), Raynaud phenomenon (OR = 7.30; P = .034), and malignancy (OR = 350.77; P = .013). The risk of malignancy was also associated with older age (OR 1.31; P = .012), and male patients were associated with a higher risk of fever. For MSAs, anti-aminoacyl-tRNA synthetase antibodies were associated with ILD, antinuclear antibody were associated with a lower risk of arthritis, anti-transcription intermediary factor 1-gamma antibodies were associated with milder symptoms of muscle weakness, anti-Ku antibodies were associated with overlap diagnosis of systemic lupus erythematosus, and anti-Ro52 antibodies were associated with the development of Raynaud phenomenon and Sjögren syndrome.MSAs and overlap diagnosis of systemic sclerosis were significantly associated with clinical phenotypes of DM and PM. Physicians should be vigilant for malignancy in older DM and PM patients with overlap diagnosis of systeic sclerosis. The possibility of developing ILD in patients with overlap diagnosis of systemic sclerosis or serum positivity of anti-aminoacyl-tRNA synthetase antibodies should be considered.

Intracellular vesicle clusters are organelles that synthesize extracellular vesicle-associated cargo proteins in yeast.

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

Which Fruits and Vegetables Should Be Excluded from a Low-Salicylate Diet? An Analysis of Salicylic Acid in Foodstuffs in Taiwan.

BACKGROUND: Eliminating pseudoallergens is an important element of managing chronic spontaneous urticaria (CSU). Salicylic acid (SA) is a primary pseudoallergen in plant-based foodstuffs. Current dietary recommendations are not applicable in East Asia because data on the SA content of many vegetables and fruits commonly consumed in this region are lacking. METHODS: We therefore determined the concentration of free SA in 79 popular vegetables and fruits frequently consumed in Taiwan using gas chromatography-mass spectrometry. RESULTS: The SA content ranged from 0.09 to 2.3 mg/kg in the fresh vegetables examined, and from 0.01 to 0.48 mg/kg in the fruits. CONCLUSIONS: Data regarding the SA content of East Asian vegetables and fruits could help CSU patients limit their pseudoallergen consumption.

The Shape of Vesicle-Containing Organelles Is Critical for Their Functions in Vesicle Endocytosis.

Exosomes are small vesicles secreted by a variety of cell types under physiological and pathological conditions. When Saccharomyces cerevisiae are grown in low glucose, small vesicles carrying more than 300 proteins with diverse biological functions are secreted. Upon glucose addition, secreted vesicles are endocytosed that requires cup-shaped organelles containing the major eisosome protein Pil1p at the rims. We aim to identify genes that regulate the function of cup-shaped organelles in vesicle endocytosis. In cells lacking either VID27 or VID21, Pil1p distribution was altered and cup-shaped organelles became elongated with narrower openings. Change in shape reduced the number of vesicles in the deeper areas and impaired vesicle endocytosis. Vid21p and Vid27p were localized to vesicle clusters and interacted with other Vid proteins. In the absence of these genes, these vesicles failed to aggregate and were secreted. Vid21p and Vid27p are required for the aggregation and retention of vesicles that contain Vid proteins in the cytoplasm. Increased vesicles near the plasma membrane in mutant strains correlate with an increased Pil1p movement resulting in the fusion of cup-shaped organelles. We conclude that the shape of vesicle-containing organelles is critical for their functions in vesicle endocytosis.

Vps15p regulates the distribution of cup-shaped organelles containing the major eisosome protein Pil1p to the extracellular fraction required for endocytosis of extracellular vesicles carrying metabolic enzymes.

BACKGROUND INFORMATION: Exosomes are small vesicles secreted from virtually every cell from bacteria to humans. Saccharomyces cerevisiae is a model system to study trafficking of small vesicles in response to changes in the environment. When yeast cells are grown in low glucose, vesicles carrying gluconeogenic enzymes are present as free vesicles and aggregated clusters in the cytoplasm. These vesicles are also secreted into the periplasm and account for more than 90% of total extracellular organelles, while less than 10% are larger 100-300 nm structures with unknown functions. When glucose is added to glucose-starved cells, secreted vesicles are endocytosed and then targeted to the vacuole. Recent secretomic studies indicated that more than 300 proteins involved in diverse biological functions are secreted during glucose starvation and endocytosed during glucose re-feeding. We hypothesised that extracellular vesicles are internalised using novel mechanisms independent of clathrin-mediated endocytosis. RESULTS: Our results showed that vesicles carrying metabolic enzymes were endocytosed at a fast rate, whereas vesicles carrying the heat shock protein Ssa1p were endocytosed at a slow rate. The PI3K regulator Vps15p is critical for the fast internalisation of extracellular vesicles. VPS15 regulates the distribution of the 100-300 nm organelles that contain the major eisosome protein Pil1p to the extracellular fraction. These Pil1p-containing structures were purified and showed unique cup-shape with their centres deeper than the peripheries. In the absence of VPS15, PIL1 or when PIL1 was mutated, the 100-300 nm structures were not observed in the extracellular fraction and the rapid internalisation of vesicles was impaired. CONCLUSIONS: We conclude that VPS15 regulates the distribution of the 100-300 nm Pil1p-containing organelles to the extracellular fraction required for fast endocytosis of vesicles carrying metabolic enzymes. This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm. SIGNIFICANCE: This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm.

Yeast as a Model System to Study Trafficking of Small Vesicles Carrying Signal-less Proteins In and Out of the Cell.

Exosomes are small vesicles that are released from a variety of cells and are involved in cell-to-cell communication. In humans, exosomes are detected in the plasma, urine, saliva, and cerebrospinal fluid. These vesicles carry multiple cargo proteins, as well as microRNA that affect the transcription of target genes. During cancer progression, secretion of exosomes increases. As such, proteins and microRNAs released from exosomes in cancer patients can be used as biomarkers for cancer diagnosis and progression. Yeast is an excellent model system to study trafficking of small vesicles in and out of the cells. When yeast cells are grown in low glucose, small vesicles transport gluconeogenic enzymes to different locations. In the cytoplasm, they exist as free vesicles and aggregated clusters. They are also secreted and account for more than 90% of total organelles in the extracellular fraction. When glucose is added to prolongedstarved cells, extracellular vesicles are endocytosed resulting in a dramatic decrease in vesicles in the extracellular fraction and the appearance of vesicle clusters in the cytoplasm. Internalization also leads to a rapid decline in protein levels for vesicle-associated proteins in the extracellular fraction. Using large-scale proteomics/secretomics, more than 300 proteins that were secreted into the extracellular fraction/periplasm were identified. They showed distribution patterns similar to gluconeogenic enzymes. They were secreted during glucose starvation and their levels in the extracellular fraction decreased following glucose re-feeding. Multiple techniques have been used to study the biogenesis, clustering, secretion, and internalization of vesicles in yeast.

Exocytosis and Endocytosis of Small Vesicles across the Plasma Membrane in Saccharomyces cerevisiae.

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase, isocitrate lyase, and malate dehydrogenase, as well as the non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, are secreted into the periplasm. In the extracellular fraction, these secreted proteins are associated with small vesicles that account for more than 90% of the total number of extracellular structures observed. When glucose is added to glucose-starved cells, FBPase is internalized and associated with clusters of small vesicles in the cytoplasm. Specifically, the internalization of FBPase results in the decline of FBPase and vesicles in the extracellular fraction and their appearance in the cytoplasm. The clearance of extracellular vesicles and vesicle-associated proteins from the extracellular fraction is dependent on the endocytosis gene END3. This internalization is regulated when cells are transferred from low to high glucose. It is rapidly occurring and is a high capacity process, as clusters of vesicles occupy 10%-20% of the total volume in the cytoplasm in glucose re-fed cells. FBPase internalization also requires the VPS34 gene encoding PI3K. Following internalization, FBPase is delivered to the vacuole for degradation, whereas proteins that are not degraded may be recycled.

The endocytosis gene END3 is essential for the glucose-induced rapid decline of small vesicles in the extracellular fraction in Saccharomyces cerevisiae.

BACKGROUND: Protein secretion is a fundamental process in all living cells. Gluconeogenic enzymes are secreted when Saccharomyces cerevisiae are grown in media containing low glucose. However, when cells are transferred to media containing high glucose, they are internalized. We investigated whether or not gluconeogenic enzymes were associated with extracellular vesicles in glucose-starved cells. We also examined the role that the endocytosis gene END3 plays in the internalization of extracellular proteins/vesicles in response to glucose addition. METHODS: Transmission electron microscopy was performed to determine the presence of extracellular vesicles in glucose-starved wild-type cells and the dynamics of vesicle transport in cells lacking the END3 gene. Proteomics was used to identify extracellular proteins that associated with these vesicles. RESULTS: Total extracts prepared from glucose-starved cells consisted of about 95% small vesicles (30-50 nm) and 5% large structures (100-300 nm). The addition of glucose caused a rapid decline in small extracellular vesicles in wild-type cells. However, most of the extracellular vesicles were still observed in cells lacking the END3 gene following glucose replenishment. Proteomics was used to identify 72 extracellular proteins that may be associated with these vesicles. Gluconeogenic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase, as well as non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, were distributed in the vesicle-enriched fraction in total extracts prepared from cells grown in low glucose. Distribution of these proteins in the vesicle-enriched fraction required the integrity of the membranes. When glucose was added to glucose-starved wild-type cells, levels of extracellular fructose-1,6-bisphosphatase, malate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin A were reduced. In contrast, in cells lacking the END3 gene, levels of these proteins in the extracellular fraction remained high. CONCLUSION: The END3 gene is required for the rapid decline of extracellular proteins and vesicles in response to glucose addition.

Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

BACKGROUND: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. RESULTS: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. CONCLUSIONS: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted during prolonged glucose starvation and is internalized following glucose re-feeding via the non-classical secretory and internalizing pathways in Saccharomyces cerevisiae.

In Saccharomyces cerevisia, the key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted into the periplasm during prolonged glucose starvation and is internalized into Vid/endosomes following glucose re-feeding. Fructose-1,6-bisphosphatase does not contain signal sequences required for the classical secretory and endocytic pathways. Hence, the secretion and internalization are mediated via the non-classical pathways.

Vid28 protein is required for the association of vacuole import and degradation (Vid) vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Gluconeogenic enzymes are induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway is linked to the nonclassical secretory and internalizing pathways. In prolonged starved cells, substantial amounts of the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) are in the extracellular fraction (periplasm). However, when glucose is added to glucose-starved cells, levels of extracellular FBPase decrease rapidly. Ultrastructural studies indicate that FBPase is in Vid/endosomes following glucose addition, suggesting that FBPase is internalized in response to glucose refeeding. Under the same conditions, the majority of Vid vesicle proteins are in the intracellular fraction. In yeast, actin polymerization is involved in endocytosis. Vid vesicles associate with actin patches initially, and they dissociate later. Here, we show that VID28 plays a critical role in the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction. Vid28p was distributed to Vid vesicles and interacted with other Vid vesicle proteins. Vid28p contains an Armadillo (ARM) domain required for FBPase degradation. When VID28 was deleted or when the ARM domain was mutated, Vid vesicles failed to co-localize with actin patches, and Vid vesicle proteins appeared in the extracellular fraction. We suggest that the ARM domain is required for the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Fructose-1,6-bisphosphatase, Malate Dehydrogenase, Isocitrate Lyase, Phosphoenolpyruvate Carboxykinase, Glyceraldehyde-3-phosphate Dehydrogenase, and Cyclophilin A are secreted in Saccharomyces cerevisiae grown in low glucose.

Our previous studies demonstrated that the key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted when Saccharomyces cerevisiae are starved of glucose for a prolonged period of time. In this study, we showed that malate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin A are also secreted in glucose-starved cells. Thus, both gluconeogenic and non-gluconeogenic enzymes are secreted via the non-classical pathway.

Vps34p is required for the decline of extracellular fructose-1,6-bisphosphatase in the vacuole import and degradation pathway.

When Saccharomyces cerevisiae are starved of glucose for a prolonged period of time, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase are induced. However, when glucose is added to prolonged-starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway merges with the endocytic pathway to remove intracellular and extracellular proteins simultaneously. Ultrastructural and cell extraction studies indicate that substantial amounts of FBPase were in the extracellular fraction (periplasm) during glucose starvation. FBPase levels in the extracellular fraction decreased after glucose re-feeding in wild-type cells. The decline of FBPase in the extracellular fraction was dependent on the SLA1 and ARC18 genes involved in actin polymerization and endocytosis. Moreover, the reduction of extracellular FBPase was also dependent on the VPS34 gene. VPS34 encodes the PI3 kinase and is also required for the Vid pathway. Vps34p co-localized with actin patches in prolonged-starved cells. In the absence of this gene, FBPase and the Vid vesicle protein Vid24p associated with actin patches before and after the addition of glucose. Furthermore, high levels of FBPase remained in the extracellular fraction in the Δvps34 mutant during glucose re-feeding. When the Asn-736 residue of Vps34p was mutated and when the C-terminal 11 amino acids were deleted, mutant proteins failed to co-localize with actin patches, and FBPase in the extracellular fraction did not decrease as rapidly. We suggest that VPS34 plays a critical role in the decline of extracellular FBPase in response to glucose.

Comparative proteomic analysis of transition of saccharomyces cerevisiae from glucose-deficient medium to glucose-rich medium.

BACKGROUND: When glucose is added to Saccharomyces cerevisiae grown in non-fermentable carbon sources, genes encoding ribosomal, cell-cycle, and glycolytic proteins are induced. By contrast, genes involved in mitochondrial functions, gluconeogenesis, and the utilization of other carbon sources are repressed. Glucose also causes the activation of the plasma membrane ATPase and the inactivation of gluconeogenic enzymes and mitochondrial enzymes. The goals of this study were to use the iTRAQ-labeling mass spectrometry technique to identify proteins whose relative levels change in response to glucose re-feeding and to correlate changes in protein abundance with changes in transcription and enzymatic activities. We used an experimental condition that causes the degradation of gluconeogenic enzymes when glucose starved cells are replenished with glucose. Identification of these enzymes as being down-regulated by glucose served as an internal control. Furthermore, we sought to identify new proteins that were either up-regulated or down-regulated by glucose. RESULTS: We have identified new and known proteins that change their relative levels in cells that were transferred from medium containing low glucose to medium containing high glucose. Up-regulated proteins included ribosomal subunits, proteins involved in protein translation, and the plasma membrane ATPase. Down-regulated proteins included small heat shock proteins, mitochondrial proteins, glycolytic enzymes, and gluconeogenic enzymes. Ach1p is involved in acetate metabolism and is also down-regulated by glucose. CONCLUSIONS: We have identified known proteins that have previously been reported to be regulated by glucose as well as new glucose-regulated proteins. Up-regulation of ribosomal proteins and proteins involved in translation may lead to an increase in protein synthesis and in nutrient uptake. Down-regulation of glycolytic enzymes, gluconeogenic enzymes, and mitochondrial proteins may result in changes in glycolysis, gluconeogenesis, and mitochondrial functions. These changes may be beneficial for glucose-starved cells to adapt to the addition of glucose.

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway.

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway.

Glucose deprivation induces the synthesis of pivotal gluconeogenic enzymes such as fructose-1,6-bisphosphatase, malate dehydrogenase, phosphoenolpyruvate carboxykinase and isocitrate lyase in Saccharomyces cerevisiae. However, following glucose replenishment, these gluconeogenic enzymes are inactivated and degraded. Studies have characterized the mechanisms by which these enzymes are inactivated in response to glucose. The site of degradation of these proteins has also been ascertained to be dependent on the duration of starvation. Glucose replenishment of short-term starved cells results in these proteins being degraded in the proteasome. In contrast, addition of glucose to cells starved for a prolonged period results in these proteins being degraded in the vacuole. In the vacuole dependent pathway, these proteins are sequestered in specialized vesicles termed vacuole import and degradation (Vid). These vesicles converge with the endocytic pathway and deliver their cargo to the vacuole for degradation. Recent studies have identified that internalization, as mediated by actin polymerization, is essential for delivery of cargo proteins to the vacuole for degradation. In addition, components of the target of rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissociates from cargo proteins following glucose replenishment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.

The TOR complex 1 is distributed in endosomes and in retrograde vesicles that form from the vacuole membrane and plays an important role in the vacuole import and degradation pathway.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.