RESUMEN
Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by hexokinases or glucokinases. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2, and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, yeast Hxk2 has been proposed to shuttle into the nucleus in glucose-replete conditions, where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve its role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 and requires an N-terminal nuclear localization sequence (NLS). We used high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. Countering previous yeast studies, we find that Hxk2 is largely excluded from the nucleus under glucose-replete conditions but is retained in the nucleus under glucose-limiting conditions. We find that the Hxk2 N-terminus does not contain an NLS but instead is necessary for nuclear exclusion and regulating multimerization. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Alanine substation at nearby lysine 13 affects dimerization and maintenance of nuclear exclusion in glucose-replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 regulates Hxk2 localization. RNAseq analyses of the yeast transcriptome dispels the idea that Hxk2 moonlights as a transcriptional regulator of glucose repression, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose-replete and limiting conditions. Our studies define a new model of cis- and trans-acting regulators of Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, which aligns well with the nuclear regulation of mammalian orthologs. Our results lay the foundation for future studies of Hxk2 nuclear activity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucosa/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/genética , Serina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Rapidly proliferating cells growing by glucose fermentation must first transport glucose into the cell. Both budding yeast and human tumor cells utilize members of a conserved family of glucose transporters. In this issue of The EMBO Journal, Stahl et al (2019) reveal that budding yeast cells confer a growth advantage to their daughters using a novel mechanism, the asymmetric distribution to the daughter cell of the mRNA for a specific glucose transporter.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa , Proteínas de Saccharomyces cerevisiae , Glucosa , Humanos , ARN Mensajero , Saccharomyces cerevisiae/genéticaRESUMEN
Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.
Asunto(s)
Desoxiglucosa , Hexoquinasa , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND INFORMATION: Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. RESULTS: We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. CONCLUSIONS: The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. SIGNIFICANCE: To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.
Asunto(s)
Arrestinas , Proteínas de Saccharomyces cerevisiae , Arrestinas/metabolismo , Endocitosis , Homeostasis , Fosfatos de Inositol , Fosfolípidos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.
Asunto(s)
Metabolismo Energético/genética , Glucosa/metabolismo , Hexoquinasa/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucosa/efectos adversos , Desoxiglucosa/farmacología , Endocitosis/efectos de los fármacos , Endocitosis/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Mutación/genética , Proteína Fosfatasa 1/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacos , Secuenciación Completa del GenomaRESUMEN
Yeast and cancer cells are metabolically similar as they use fermentation of glucose as a primary means of generating energy. Reliance on glucose fermentation makes both of these cell types highly sensitive to the toxic glucose analog, 2-deoxyglucose. Here we review the cellular and metabolic pathways that play a role in 2-deoxyglucose sensitivity and discuss how the modifications to these pathways result in acquisition of 2-deoxyglucose resistance. Insights gained from genetic and proteomic studies in yeast provide new ideas for the design of combinatorial therapies for cancer treatment.
Asunto(s)
Daño del ADN/genética , Desoxiglucosa/genética , Endocitosis/genética , Proteómica , Glucosa/genética , Glucosa/metabolismo , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important for ROMK regulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMK and establish that components in this pathway modulate channel activity.
Asunto(s)
Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Vacuolas/metabolismo , Células HEK293 , Humanos , Mutación , Canales de Potasio de Rectificación Interna/genética , Transporte de ProteínasRESUMEN
Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.
Asunto(s)
Arrestina/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Modelos Biológicos , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arrestina/genética , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. Recent studies reveal a novel role for AMPK in the regulation of glucose and other carbohydrates flux by controlling the endocytosis of transporters. The first step in glucose metabolism is glucose uptake, a process mediated by members of the GLUT/SLC2A (glucose transporters) or HXT (hexose transporters) family of twelve-transmembrane domain glucose transporters in mammals and yeast, respectively. These proteins are conserved from yeast to humans, and multiple transporters-each with distinct kinetic properties-compete for plasma membrane occupancy in order to enhance or limit the rate of glucose uptake. During growth in the presence of alternative carbon sources, glucose transporters are removed and replaced with the appropriate transporter to help support growth in response to this environment. New insights into the regulated protein trafficking of these transporters reveal the requirement for specific α-arrestins, a little-studied class of protein trafficking adaptor. A defining feature of the α-arrestins is that each contains PY-motifs, which can bind to the ubiquitin ligases from the NEDD4/Rsp5 (Neural precursor cell Expressed, Developmentally Down-regulated 4 and Reverses Spt- Phenotype 5, respectively) family. Specific association of α-arrestins with glucose and carbohydrate transporters is thought to bring the ubiquitin ligase in close proximity to its membrane substrate, and thereby allows the membrane cargo to become ubiquitinated. This ubiquitination in turn serves as a mark to stimulate endocytosis. Recent results show that AMPK phosphorylation of the α-arrestins impacts their abundance and/or ability to stimulate carbohydrate transporter endocytosis. Indeed, AMPK or glucose limitation also controls α-arrestin gene expression, adding an additional layer of complexity to this regulation. Here, we review the recent studies that have expanded the role of AMPK in cellular metabolism to include regulation of α-arrestin-mediated trafficking of transporters and show that this mechanism of regulation is conserved over the ~150 million years of evolution that separate yeast from man.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Arrestina/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Animales , Arrestina/química , Sitios de Unión , Endocitosis , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Humanos , Transporte de Proteínas , UbiquitinaciónRESUMEN
Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood. For CME in yeast, particular α-arrestins contribute to recognition of specific cargos and promote their ubiquitylation by recruiting the E3 ubiquitin protein ligase Rsp5. Here, we show that the same α-arrestin-cargo pairs promote internalization through the CIE pathway by interacting with CIE components. Notably, neither expression of Rsp5 nor its binding to α-arrestins is required for CIE. Thus, α-arrestins are important for cargo selection in both the CME and CIE pathways, but function by distinct mechanisms in each pathway.
Asunto(s)
Arrestinas/metabolismo , Transporte Biológico/genética , Clatrina/metabolismo , Endocitosis/genética , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Membrana Celular , Saccharomyces cerevisiae/metabolismoRESUMEN
Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.
Asunto(s)
Arrestinas/metabolismo , Calcineurina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arrestinas/química , Dominio Catalítico , Cromatografía Liquida , Endocitosis , Humanos , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Fenotipo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Especificidad por Sustrato , Ubiquitinación , Vacuolas/metabolismoRESUMEN
Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.
RESUMEN
Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.
Asunto(s)
Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Anticuerpos de Cadena Única/metabolismo , Endocitosis/fisiología , Colorantes Fluorescentes/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Fluorescentes Verdes/metabolismo , Colorantes de RosanilinaRESUMEN
Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/biosíntesis , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico , Vacuolas/metabolismoRESUMEN
Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.
Asunto(s)
Curcumina/farmacología , Quelantes del Hierro/farmacología , Hierro/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Transporte Biológico , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Cobre/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Hierro/análisis , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109137) describe a yeast endosomal recycling pathway that gives proteins a second chance to return to the plasma membrane.
Asunto(s)
Endosomas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Transporte Biológico , Membrana Celular/metabolismoRESUMEN
Nutrient supply dictates cell signaling changes, which in turn regulate membrane protein trafficking. To better exploit nutrients, cells relocalize membrane transporters via selective protein trafficking. Key in this reshuffling are the α-arrestins, selective protein trafficking adaptors conserved from yeast to man. α-Arrestins bind membrane proteins, controlling the ubiquitination and endocytosis of many transporters. To prevent the spurious removal of membrane proteins, α-arrestin-mediated endocytosis is kept in check through phospho-inhibition. This phospho-regulation is complex, with up to 87 phospho-sites on a single α-arrestin and many kinases/phosphatases targeting α-arrestins. To better define the signaling pathways controlling paralogous α-arrestins, Aly1 and Aly2, we screened the kinase and phosphatase deletion (KinDel) library, which is an array of all non-essential kinase and phosphatase yeast deletion strains, for modifiers of Aly-mediated phenotypes. We identified many Aly regulators, but focused our studies on the TORC1 kinase, a master regulator of nutrient signaling across eukaryotes. We found that TORC1 and its signaling effectors, the Sit4 protein phosphatase and Npr1 kinase, regulate the phosphorylation and stability of Alys. When Sit4 is lost, Alys are hyperphosphorylated and destabilized in an Npr1-dependent manner. These findings add new dimensions to our understanding of TORC1 regulation of α-arrestins and have important ramifications for cellular metabolism.
Asunto(s)
Arrestinas , Proteínas de Saccharomyces cerevisiae , Arrestina/metabolismo , Arrestinas/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteína Fosfatasa 2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Potassium (K+) homeostasis is tightly regulated for optimal cell and organismal health. Failure to control potassium balance results in disease, including cardiac arrythmias and developmental disorders. A family of inwardly rectifying potassium (Kir) channels helps cells maintain K+ levels. Encoded by KCNJ genes, Kir channels are comprised of a tetramer of Kir subunits, each of which contains two-transmembrane domains. The assembled Kir channel generates an ion selectivity filter for K+ at the monomer interface, which allows for K+ transit. Kir channels are found in many cell types and influence K+ homeostasis across the organism, impacting muscle, nerve and immune function. Kir2.1 is one of the best studied family members with well-defined roles in regulating heart rhythm, muscle contraction and bone development. Due to their expansive roles, it is not surprising that Kir mutations lead to disease, including cardiomyopathies, and neurological and metabolic disorders. Kir malfunction is linked to developmental defects, including underdeveloped skeletal systems and cerebellar abnormalities. Mutations in Kir2.1 cause the periodic paralysis, cardiac arrythmia, and developmental deficits associated with Andersen-Tawil Syndrome. Here we review the roles of Kir family member Kir2.1 in maintaining K+ balance with a specific focus on our understanding of Kir2.1 channel trafficking and emerging roles in development and disease. We provide a synopsis of the vital work focused on understanding the trafficking of Kir2.1 and its role in development.
RESUMEN
Recent evidence demonstrates that novel protein-coding genes can arise de novo from non-genic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of non-genic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Here, we systematically characterize how these de novo emerging coding sequences impact fitness in budding yeast. Disruption of emerging sequences is generally inconsequential for fitness in the laboratory and in natural populations. Overexpression of emerging sequences, however, is enriched in adaptive fitness effects compared to overexpression of established genes. We find that adaptive emerging sequences tend to encode putative transmembrane domains, and that thymine-rich intergenic regions harbor a widespread potential to produce transmembrane domains. These findings, together with in-depth examination of the de novo emerging YBR196C-A locus, suggest a novel evolutionary model whereby adaptive transmembrane polypeptides emerge de novo from thymine-rich non-genic regions and subsequently accumulate changes molded by natural selection.
Asunto(s)
Evolución Molecular , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Timina , Factor de Transcripción TFIID/genética , Adaptación Biológica/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación Fúngica de la Expresión Génica , Aptitud Genética , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Sistemas de Lectura Abierta , Dominios Proteicos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Transcription by RNA polymerase II is impeded by the nucleosomal organization of DNA; these negative effects are modulated at several stages of nucleosomal DNA transcription by FACT, a heterodimeric transcription factor. At promoters, FACT facilitates the binding of TATA-binding factor, while during transcription elongation FACT mediates the necessary destabilization of nucleosomes and subsequent restoration of nucleosome structure in the wake of the transcription elongation complex. Altered FACT activity can impair the fidelity of transcription initiation and affect transcription patterns. Using reporter genes we have identified new mutant versions of the Spt16 subunit of yeast FACT with dominant negative effects on the fidelity of transcription initiation. Two of these spt16 mutant alleles also affect cell integrity. Cells relying on these spt16 mutant alleles display sorbitol-remediated temperature sensitivity, altered sensitivity to detergent, and abnormal morphologies, and are further inhibited by the ssd1-d mutation. The overexpression of components of protein kinase C (Pkc1) signaling diminishes this spt16 ssd1-d temperature sensitivity, whereas gene deletions eliminating components of Pkc1 signaling further impair these spt16 mutant cells. Thus, the FACT subunit Spt16 and Pkc1 signaling have an overlapping essential function, with an unexpected role for FACT in the maintenance of cell integrity.