The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Bruno Andre - One of the best experts on this subject based on the ideXlab platform.
-
Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases
Molecular and Cellular Biology, 2014Co-Authors: Kassem Ghaddar, Ahmad Merhi, Elie Saliba, Eva-maria Krammer, Martine Prévost, Bruno AndreAbstract:Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid Permease (Gap1) and the arginine-specific Permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on Permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the Permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the Permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the Permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.
-
stress conditions promote yeast gap1 Permease ubiquitylation and down regulation via the arrestin like bul and aly proteins
Journal of Biological Chemistry, 2014Co-Authors: Myriam Crapeau, Ahmad Merhi, Bruno AndreAbstract:Gap1, the yeast general amino acid Permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other Permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from Permease degradation.
-
Converting the Yeast Arginine Can1 Permease to a Lysine Permease
The Journal of biological chemistry, 2014Co-Authors: Kassem Ghaddar, Bruno Andre, Eva-maria Krammer, Natalija Mihajlovic, Sylvain Brohée, Martine PrévostAbstract:Amino acid uptake in yeast cells is mediated by about 16 plasma membrane Permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid Permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific Permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like Permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 Permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.
-
signals and mechanisms controlling the ubiquitylation and down regulation of the yeast general amino acid Permease
2011Co-Authors: Ahmad Merhi, Bruno AndreAbstract:Cell surface transport proteins play a crucial role in all cells, from unicellular organisms to mammals, by conferring to the plasma membrane selective permeability to a wide range of ions and small molecules. The activity of these proteins is very often regulated by controlling their amount at the plasma membrane where they are removed by means of selective endocytosis in response to signals and changes in the environment.One of the membrane proteins of the yeast Saccharomyces cerevisiae whose regulation has been extensively studied is the general amino acid Permease. Previous studies on Gap1 and other yeast Permeases revealed that ubiquitin plays a key role in the membrane trafficking of these proteins by providing a signal that triggers their internalization in endocytic vesicles and that promote their sorting into intra-endosomal vesicles for subsequent delivery into the lumen of the vacuole, the lysosome of yeast. In the first part of this work, we report the isolation of 64 mutant forms of the Gap1 protein and their exploitation in a systematic functional study of the predicted intracellular regions of the Permease. The phenotypic analysis of these mutants revealed an important role of certain amino acid sequences in the (i) transport of the Permease through the secretory pathway (ii) intrinsic activity of the Permease at the plasma membrane (iii) stability of the protein at the cell surface (iv) sorting of the protein into intra-endosomal vesicles. Further investigation of some of these mutants allowed us to unravel an original mechanism for the degradation of the Permease that is independent of its ubiquitylation.In the second part of the work, we used yet other Gap1 mutants to study the signals and pathways inducing the ubiquitylation and endocytosis of the Permease. Also, we further investigated the molecular mechanisms inducing Gap1 ubiquitylation. All these results together allow us to better understand the mechanisms controlling the ubiquitin dependent down-regulation of plasma membrane proteins.
-
The ubiquitin code of yeast Permease trafficking
Trends in Cell Biology, 2010Co-Authors: Elsa Lauwers, Rosine Haguenauer-tsapis, Zoi Erpapazoglou, Bruno AndreAbstract:Yeast Permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiological control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent studies have uncovered the role of specific adaptors for recruiting the Rsp5 ubiquitin ligase to these proteins. In addition, the role of ubiquitin at different trafficking steps including early endocytosis, sorting into the multivesicular body (MVB) pathway and Golgi-to-endosome transit is now becoming clear. In particular, K63-linked ubiquitin chains now emerge as a specific signal for protein sorting into the MVB pathway. A complete view of the ubiquitin code governing yeast Permease trafficking might not be far off.
Johanowen De Craene - One of the best experts on this subject based on the ideXlab platform.
-
the npr1 kinase controls biosynthetic and endocytic sorting of the yeast gap1 Permease
Journal of Biological Chemistry, 2001Co-Authors: Johanowen De Craene, Oriane Soetens, Bruno AndreAbstract:Membrane trafficking of the general amino acid Permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH(4)(+), a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the Permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(ts) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH(4)(+) is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH(4)(+). Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other Permeases. We propose that these kinases also function as regulators of Permease trafficking.
-
The Npr1 Kinase Controls Biosynthetic and Endocytic Sorting of the Yeast Gap1 Permease
Journal of Biological Chemistry, 2001Co-Authors: Johanowen De Craene, Oriane Soetens, Bruno AndreAbstract:Membrane trafficking of the general amino acid per-mease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH 4 , a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquiti-nation of the Permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1 ts mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH 4 is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1 mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH 4. Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other Permeases. We propose that these kinases also function as regulators of Permease trafficking.
-
amino acid signaling in saccharomyces cerevisiae a Permease like sensor of external amino acids and f box protein grr1p are required for transcriptional induction of the agp1 gene which encodes a broad specificity amino acid Permease
Molecular and Cellular Biology, 1999Co-Authors: Ismail Iraqui, Florent Bernard, Johanowen De Craene, Antonio Urrestarazu, Eckhard Boles, Stephan Vissers, Bruno AndreAbstract:The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid Permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid Permease. Total noninduction of the AGP1 gene in the ssy1Delta mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid Permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid Permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.
-
Amino Acid Signaling in Saccharomyces cerevisiae: a Permease- Like Sensor of External Amino Acids and F-Box Protein Grr1p Are Required for Transcriptional Induction of the AGP1 Gene, Which Encodes a Broad-Specificity Amino Acid Permease
Molecular and Cellular Biology, 1999Co-Authors: Ismail Iraqui, Florent Bernard, Johanowen De Craene, Antonio Urrestarazu, Eckhard Boles, Stephan Vissers, Bruno AndreAbstract:The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid Permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid Permease. Total nonin-duction of the AGP1 gene in the ssy1 mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys 6-Zn 2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCF Grr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid Permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid Permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.
Cécile Wandersman - One of the best experts on this subject based on the ideXlab platform.
-
Functional differences between heme Permeases: Serratia marcescens HemTUV Permease exhibits a narrower substrate specificity (restricted to heme) than the Escherichia coli DppABCDF peptide-heme Permease.
Journal of Bacteriology, 2008Co-Authors: Sylvie Létoffé, Philippe Delepelaire, Cécile WandersmanAbstract:Serratia marcescens hemTUV genes encoding a potential heme Permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide Permease responsible for heme, dipeptide, and delta-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp Permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp Permease for ALA uptake, indicating that the HemTUV Permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp Permease, HemTUV Permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV Permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific Permeases different from the Dpp peptide/heme Permease characterized in E. coli.
-
Functional Differences between Heme Permeases: Serratia marcescens HemTUV Permease Exhibits a Narrower Substrate Specificity (Restricted to Heme) Than the Escherichia coli DppABCDF Peptide-Heme Permease
Journal of bacteriology, 2008Co-Authors: Sylvie Létoffé, Philippe Delepelaire, Cécile WandersmanAbstract:Serratia marcescens hemTUV genes encoding a potential heme Permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide Permease responsible for heme, dipeptide, and δ-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp Permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp Permease for ALA uptake, indicating that the HemTUV Permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp Permease, HemTUV Permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV Permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific Permeases different from the Dpp peptide/heme Permease characterized in E. coli.
Arnold J. M. Driessen - One of the best experts on this subject based on the ideXlab platform.
-
pcmtr an aromatic and neutral aliphatic amino acid Permease of penicillium chrysogenum
Biochimica et Biophysica Acta, 2004Co-Authors: Hein Trip, Melchior E Evers, Arnold J. M. DriessenAbstract:The gene encoding an aromatic and neutral aliphatic amino acid Permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The Permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid Permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type Permeases are not widely distributed among fungi, suggesting a specialized function.
-
cloning and characterization of an aromatic amino acid and leucine Permease of penicillium chrysogenum
Biochimica et Biophysica Acta, 2002Co-Authors: Hein Trip, Melchior E Evers, Wil N. Konings, Arnold J. M. DriessenAbstract:Abstract The gene encoding the amino acid Permease ArlP ( Ar omatic and l eucine P ermease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid Permeases. The cDNA clone was used for expression of the Permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid Permease Gap1. Upon overexpression, an increase in the uptake of l -tyrosine, l -phenylalanine, l -tryptophan and l -leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched β-carbon atom.
-
Cloning and characterization of an aromatic amino acid and leucine Permease of Penicillium chrysogenum
Biochimica et biophysica acta, 2002Co-Authors: Hein Trip, Melchior E Evers, Wil N. Konings, Arnold J. M. DriessenAbstract:The gene encoding the amino acid Permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid Permeases. The cDNA clone was used for expression of the Permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid Permease Gap1. Upon overexpression, an increase in the uptake of L-tyrosine, L-phenylalanine, L-tryptophan and L-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched beta-carbon atom.
-
Sulfate Transport in Penicillium chrysogenum : Cloning and Characterization of the sutA and sutB Genes
1999Co-Authors: Mart Van De Kamp, Wil N. Konings, Arnold J. M. Driessen, Geoffrey Turner, Enrica Pizzinini, Arnold Vos, Ted R. Van Der Lende, Theo A. Schuurs, Roger W. NewbertAbstract:In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate Permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate Permease involved in sulfate uptake by P. chrysogenum.
Rosine Haguenauer-tsapis - One of the best experts on this subject based on the ideXlab platform.
-
The ubiquitin code of yeast Permease trafficking
Trends in Cell Biology, 2010Co-Authors: Elsa Lauwers, Rosine Haguenauer-tsapis, Zoi Erpapazoglou, Bruno AndreAbstract:Yeast Permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiological control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent studies have uncovered the role of specific adaptors for recruiting the Rsp5 ubiquitin ligase to these proteins. In addition, the role of ubiquitin at different trafficking steps including early endocytosis, sorting into the multivesicular body (MVB) pathway and Golgi-to-endosome transit is now becoming clear. In particular, K63-linked ubiquitin chains now emerge as a specific signal for protein sorting into the MVB pathway. A complete view of the ubiquitin code governing yeast Permease trafficking might not be far off.
-
Uracil-Induced Down-Regulation of the Yeast Uracil Permease
Journal of Bacteriology, 1999Co-Authors: Karin Séron, Marie-odile Blondel, Rosine Haguenauer-tsapis, Christiane VollandAbstract:In Saccharomyces cerevisiae the FUR4-encoded uracil Permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil Permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing Permease by promoting high efficiency of ubiquitination of the Permease that signals its internalization. A direct binding of intracellular uracil to the Permease is possibly involved in this feedback regulation, as the behavior of the Permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the Permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate Permease to prevent buildup of excess intracellular uracil-derived nucleotides.
-
A PEST-like sequence mediates phosphorylation and efficient ubiquitination of yeast uracil Permease.
Molecular and cellular biology, 1998Co-Authors: Christelle Marchal, Rosine Haguenauer-tsapis, Danièle Urban-grimalAbstract:Uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific Permease encoded by the FUR4 gene. Uracil Permease located at the cell surface is subject to two covalent modifications: phosphorylation and ubiquitination. The ubiquitination step is necessary prior to Permease endocytosis and subsequent vacuolar degradation. Here, we demonstrate that a PEST-like sequence located within the cytoplasmic N terminus of the protein is essential for uracil Permease turnover. Internalization of the transporter was reduced when some of the serines within the region were converted to alanines and severely impaired when all five serines within the region were mutated or when this region was absent. The phosphorylation and degree of ubiquitination of variant Permeases were inversely correlated with the number of serines replaced by alanines. A serine-free version of this sequence was very poorly phosphorylated, and elimination of this sequence prevented ubiquitination. Thus, it appears that the serine residues in the PEST-like sequence are required for phosphorylation and ubiquitination of uracil Permease. A PEST-like sequence in which the serines were replaced by glutamic acids allowed efficient Permease turnover, suggesting that the PEST serines are phosphoacceptors.
-
Endocytosis and degradation of the yeast uracil Permease under adverse conditions.
The Journal of biological chemistry, 1994Co-Authors: Christiane Volland, Danièle Urban-grimal, G Géraud, Rosine Haguenauer-tsapisAbstract:Yeast uracil Permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to be rather stable in growing cells, but its turnover rate (half-life of about 7 h) was much faster than that of most yeast proteins. Several adverse conditions triggered the rapid degradation of uracil Permease, and so a loss of uracil uptake. Turnover was rapid when yeast cells were starved of either nitrogen, phosphate, or carbon, and as they approached the stationary growth phase. Rapid Permease degradation was also promoted by the inhibition of protein synthesis. The degradation of uracil Permease in response to several stresses was strikingly slower in the two mutants, end3 and end4, that are deficient in the internalization step of receptor-mediated endocytosis. Thus, internalization is the first step in the Permease degradative pathway. Uracil Permease is degraded in the vacuole, since pep4 mutant cells lacking vacuolar protease activities accumulated large amounts of uracil Permease, which was located within the vacuole by immunofluorescence. We have yet to determine whether adverse conditions enhance Permease endocytosis and subsequent degradation or divert internalized uracil Permease from a recycling to a degradative pathway.
