RESUMEN
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
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.
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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
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.
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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
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.
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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
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
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
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
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.
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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
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.
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Proteínas Facilitadoras del Transporte de la Glucosa , Proteínas de Saccharomyces cerevisiae , Glucosa , Humanos , ARN Mensajero , 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.
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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
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.
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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
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.
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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
Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.
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Endocitosis , Transporte de Proteínas , Saccharomycetales , Microscopía Fluorescente , VacuolasRESUMEN
Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which ß subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Álcalis/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/enzimología , Estrés Fisiológico/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Secuencia Conservada , Activación Enzimática , Glucosa/farmacología , Histidina/metabolismo , Isoenzimas/metabolismo , Cinética , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Subunidades de Proteína/química , Transporte de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
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.
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Arrestinas/metabolismo , Transporte Biológico/genética , Clatrina/metabolismo , Endocitosis/genética , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Membrana Celular , Saccharomyces cerevisiae/metabolismoRESUMEN
The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.