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Chris A Kaiser - One of the best experts on this subject based on the ideXlab platform.
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transport activity dependent intracellular sorting of the yeast general Amino Acid Permease
Molecular Biology of the Cell, 2011Co-Authors: Natalie E Cain, Chris A KaiserAbstract:Intracellular trafficking of the general Amino Acid Permease, Gap1p, of Saccharomyces cerevisiae is regulated by Amino Acid abundance. When Amino Acids are scarce Gap1p is sorted to the plasma membrane, whereas when Amino Acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of Amino Acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1pA297V, which does not transport basic Amino Acids, is also not regulated by these Amino Acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex Amino Acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that Amino Acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.
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activity dependent reversible inactivation of the general Amino Acid Permease
Molecular Biology of the Cell, 2006Co-Authors: April L Risinger, Natalie E Cain, Esther J Chen, Chris A KaiserAbstract:The general Amino Acid Permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring Amino Acids into yeast cells for use as a nitrogen source. Previous studies have shown that a ...
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Activity-dependent reversible inactivation of the general Amino Acid Permease.
Molecular Biology of the Cell, 2006Co-Authors: April L Risinger, Natalie E Cain, Esther J Chen, Chris A KaiserAbstract:: The general Amino Acid Permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring Amino Acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the Permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that Amino Acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of Amino Acid mixtures. Surprisingly, we also find that addition of most single Amino Acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of Amino Acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single Amino Acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of Amino Acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular Amino Acids, but does require active Amino Acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of Permease activity in response to elevated Amino Acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to Amino Acids.
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a conserved gtpase containing complex is required for intracellular sorting of the general Amino Acid Permease in yeast
Nature Cell Biology, 2006Co-Authors: Chris A KaiserAbstract:The Saccharomyces cerevisiae general Amino-Acid Permease, Gap1p, is a model for membrane proteins that are regulated by intracellular sorting according to physiological cues set by the availability of Amino Acids. Here, we report the identification of a conserved sorting complex for Gap1p, named the GTPase-containing complex for Gap1p sorting in the endosomes (GSE complex), which is required for proper sorting of Gap1p from the late endosome for eventual delivery to the plasma membrane. The complex contains two small GTPases (Gtr1p and Gtr2p) and three other proteins (Ybr077c, Ykr007w and Ltv1p) that are located in the late endosomal membrane. Importantly, Gtr2p interacts with the carboxy (C)-terminal cytosolic domain of Gap1p and a tyrosine-containing motif in this domain is necessary both to bind Gtr2p and to direct sorting of Gap1p to the plasma membrane. Together, these studies provide evidence that the GSE complex has a key role in trafficking Gap1p out of the endosome and may serve as coat proteins in this process.
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Amino Acids regulate retrieval of the yeast general Amino Acid Permease from the vacuolar targeting pathway
Molecular Biology of the Cell, 2006Co-Authors: Marta Rubiotexeira, Chris A KaiserAbstract:Intracellular sorting of the general Amino Acid Permease (Gap1p) in Saccharomyces cerevisiae depends on availability of Amino Acids such that at low Amino Acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high Permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high Amino Acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but Amino Acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas Amino Acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.
Philip S Poole - One of the best experts on this subject based on the ideXlab platform.
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rhizobium leguminosarum has a second general Amino Acid Permease with unusually broad substrate specificity and high similarity to branched chain Amino Acid transporters bra liv of the abc family
Journal of Bacteriology, 2002Co-Authors: Arthur H F Hosie, David Allaway, C S Galloway, H A Dunsby, Philip S PooleAbstract:Amino Acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general Amino Acid Permease (Aap) and the branched-chain Amino Acid Permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general Amino Acid Permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic Amino Acid transporters (HAAT), it transports a broad range of solutes, including Acidic and basic polar Amino Acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral Amino Acids (l-alanine and l-leucine). While Amino and carboxyl groups are required for transport, solutes do not have to be α-Amino Acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-Aminobutyric Acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the Amino Acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d Amino Acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general Amino Acid Permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial Amino Acid transporter.
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the general Amino Acid Permease of rhizobium leguminosarum strain 3841 is negatively regulated by the ntr system
Fems Microbiology Letters, 1997Co-Authors: D L Walshaw, Colm J Reid, Philip S PooleAbstract:Cosmid-borne and chromosomal lacZ fusions to aapJ, aapQ and aapM were used to examine the nitrogen regulation of the general Amino Acid Permease (Aap) of Rhizobium leguminosarum strain 3841. Transcription of the first gene of the operon (aapJ), which encodes the periplasmic binding protein, was 2–4-fold higher than aapQ and aapM, which encode the integral membrane proteins, under various growth conditions. This may be due to the presence of a putative stem loop in the intergenic region between aapJ and aapQ. All aap fusions were derepressed 3–5-fold after growth on glutamate as a nitrogen source, which effectively causes nitrogen limitation. An ntrC mutant was derepressed for transcription of the aap operon and had high rates of Amino Acid transport when grown on ammonia as the nitrogen source. Thus NtrC negatively regulates the aap operon, contrary to its usual role in positive gene activation. These results confirm that the aap operon is subject to complex regulation involving both transcriptional and post-transcriptional factors.
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regulation of the tca cycle and the general Amino Acid Permease by overflow metabolism in rhizobium leguminosarum
Microbiology, 1997Co-Authors: D L Walshaw, Adam Wilkinson, Mathius Mundy, Mary Smith, Philip S PooleAbstract:Summary: Mutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general Amino Acid Permease (Aap). The main class of mutant maps to sucA and sucD, which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex (sucAB). Mutation of either sucC or sucD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of sucA or sucD, the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino Acid uptake via the Aap of R. leguminosarum was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (sucD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and recluctant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by Amino Acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.
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the general l Amino Acid Permease of rhizobium leguminosarum is an abc uptake system that also influences efflux of solutes
Molecular Microbiology, 1996Co-Authors: D L Walshaw, Philip S PooleAbstract:: A general L-Amino Acid Permease (Aap) from the ABC transporter family, encoded by four genes (aapJ, Q, M, P), has been cloned and characterized in Rhizobium leguminosarum. It transports a wide range of L-Amino Acids but has a preference for those with polar side-chains. A single binding protein of broad specificity (AapJ) is required for transport of all solutes. Unusually for an ABC transporter, Aap has both high affinity for and supports high rates of solute uptake. Genes for putative Amino Acid Permeases with broad specificity for Amino Acids also exist in Escherichia coli and probably in Pseudomonas fluorescens, although the Permease from E. coli does not appear to be expressed. Aap is an active uptake system that also affects the efflux of a broad range of Amino Acids. Efflux can be measured both as the loss of an intracellular Amino Acid after the addition of an excess of a homologous or heterologous Amino Acid, and as excretion of intracellularly synthesized glutamate. Mutation of Aap prevented efflux of intracellular Amino Acids caused by the addition of an extracellular heterologous Amino Acid, while overexpression increased the rates of such efflux. Furthermore, excretion of glutamate synthesized inside the cell was reduced by 76% in an aap strain. All four gene products, including the binding protein (AapJ), appear to be needed for efflux. Aap from R. leguminosarum expressed in E. coli also promoted efflux on addition of an extracellular heterologous Amino Acid. These results indicate either that Aap regulates an efflux channel/transporter or that solute has access to the translocation pathway of Aap from both sides of the membrane.
Tomas Girbes - One of the best experts on this subject based on the ideXlab platform.
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Effect of l-azetidine 2-carboxilic Acid on the activity of the general Amino-Acid Permease from Saccharomyces cerevisiae var. ellipsoideus
Archives of Microbiology, 1991Co-Authors: Rosario Iglesias, J M Ferreras, F J Arias, R Munoz, Ma Angeles Rojo, Tomas GirbesAbstract:Addition of the l -proline analogue l -azetidine 2-carboxylic Acid to growing cultures of Saccharomyces cerevisiae var. ellipsoideus promoted fast deactivation of the general AminoAcid Permease, measured as l -valine uptake, without an immediate decrease in the growth rate. Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general AminoAcid Permease activity in analogue-free medium. Eadie-Hofstee plots of l -valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general Amino-Acid Permease located in the plasma membrane. Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.
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changes in the activity of the general Amino Acid Permease from saccharomyces cerevisiae var ellipsoideus during fermentation
Biotechnology and Bioengineering, 1990Co-Authors: R Lglesias, J M Ferreras, F J Arias, R Munoz, Tomas GirbesAbstract:: The evolution of the activity of the general Amino Acid Permease and ethanol and glucose concentrations in the medium were studied in a mild fermentation process carried out by a wine strain of Saccharomyces cerevisiae var. ellipsoideus isolated from grape musts in spontaneous fermentation. The cells displayed a reduction in the activity of the general Amino Acid Permease parallel to the increase of ethanol in the medium. This ethanol increase was not enough to promote a substantial inhibition on the total polypeptide synthesis measured as polyuridylic-Acid-directed polyphenylalanine synthesis.
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differential d glucose requirements of the general Amino Acid Permease and protein synthesis in saccharomyces cerevisiae var ellipsoideus
Cellular and Molecular Biology, 1990Co-Authors: Rosario Iglesias, J M Ferreras, F J Arias, R Munoz, M A Rojo, Tomas GirbesAbstract:: The dependence of the general AminoAcid Permease and protein synthesis on the availability of D-glucose as energy source was studied. Stimulation by the sugar was immediate once added to the cell suspensions and seems to be mediated by energy derived directly from glycolysis. The general AminoAcid Permease was saturated linearly with D-glucose whereas protein synthesis was saturated sigmoidealy requiring much higher concentration of the sugar than the general AminoAcid Permease.
Hiroshi Takagi - One of the best experts on this subject based on the ideXlab platform.
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effect of the deubiquitination enzyme gene ubp6 on the stress responsive transcription factor msn2 mediated control of the Amino Acid Permease gnp1 in yeast
Journal of Bioscience and Bioengineering, 2020Co-Authors: Noreen Suliani Binti Mat Nanyan, Daisuke Watanabe, Yukiko Sugimoto, Hiroshi TakagiAbstract:In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the Amino Acid Permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and Amino Acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic Amino Acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic Amino Acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs.
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phosphorylation of a conserved thr357 in yeast nedd4 like ubiquitin ligase rsp5 is involved in down regulation of the general Amino Acid Permease gap1
Genes to Cells, 2013Co-Authors: Toshiya Sasaki, Hiroshi TakagiAbstract:Rsp5, an essential HECT-type ubiquitin ligase, is the only yeast Saccharomyces cerevisiae member of the Nedd4 family. Rsp5 triggers the ubiquitination-dependent endocytosis of the general Amino Acid Permease Gap1 in response to a good nitrogen source. Previously, we showed that the Thr357Ala/Lys764Glu variant Rsp5 induces the constitutive inactivation of Gap1, which is mainly involved in uptake of the toxic proline analogue, l-azetidine-2-carboxylate (AZC). Here, our experimental results indicated that the Thr357Ala substitution in the substrate-recognizing WW2 domain of Rsp5 constitutively causes the down-regulation of four proline Permeases (Gap1, Put4, Agp1 and Gnp1), leading to AZC tolerance to yeast cells. In RSP5T357A cells, Gap1 was highly ubiquitinated and constantly delivered to the vacuole from the Golgi without sorting to the plasma membrane. Analyses of RSP5 mutants using antiphosphopeptide antibody suggest that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain was phosphorylated, in agreement with the in vitro result for the mouse Rsp5 orthologue. Furthermore, the phosphorylation-mimic mutant (Thr357Asp) showed strong sensitivity to AZC. From these results, we propose a possible mechanism involved in the regulation of Rsp5 activity for Gap1 down-regulation via the phosphorylation of a conserved Thr357 in the Nedd4 family.
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engineering of the yeast ubiquitin ligase rsp5 isolation of a new variant that induces constitutive inactivation of the general Amino Acid Permease gap1
Fems Yeast Research, 2009Co-Authors: Yutaka Haitani, Maiko Nakata, Toshiya Sasaki, Akiko Uchida, Hiroshi TakagiAbstract:: Rsp5 is an essential ubiquitin-protein ligase in Saccharomyces cerevisiae. We found previously that the Ala401Glu rsp5 mutant is hypersensitive to various stresses that induce protein misfolding, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. To isolate new Rsp5 variants as suppressors of the A401E mutant, PCR random mutagenesis was used in the rsp5(A401E) gene, and the mutagenized plasmid library was introduced into rsp5(A401E) cells. As a phenotypic suppressor of rsp5(A401E) cells, we isolated a quadruple variant (Thr357Ala/Glu401Gly/Lys764Glu/Glu767Gly) on a minimal medium containing the toxic proline analogue azetidine-2-carboxylate (AZC). Site-directed mutagenesis experiments showed that the rsp5(T357A/K764E) cells were much more tolerant to AZC than the wild-type cells, due to the smaller amounts of intracellular AZC. However, the T357A/K764E variant Rsp5 did not reverse the hypersensitivity of rsp5(A401E) cells to other stresses such as high growth temperature, ethanol, and freezing treatment. Interestingly, immunoblot and localization analyses indicated that the general Amino Acid Permease Gap1, which is involved in AZC uptake, was absent on the plasma membrane and degraded in the vacuole of rsp5(T357A/K764E) cells before the addition of ammonium ions. These results suggest that the T357A/K764E variant Rsp5 induces constitutive inactivation of Gap1.
Bruno Andre - One of the best experts on this subject based on the ideXlab platform.
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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.
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ubiquitin is required for sorting to the vacuole of the yeast general Amino Acid Permease gap1
Journal of Biological Chemistry, 2001Co-Authors: Oriane Soetens, Johanowen De Craene, Bruno AndreAbstract:Abstract In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general Amino Acid Permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH , a preferential source of nitrogen, is added to cells growing on proline or urea,i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1K9K16) remains fully stable at the plasma membrane after NH addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1K9 and Gap1K16 mutants, in which a single lysine is mutated, are down-regulated in response to NH although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and Andre, B. (2001) J. Biol. Chem. 276, 43939–43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Δ mutant, neosynthesized Gap1K9K16 is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Δ mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking,i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/ortrans-Golgi.
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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, Stephan Vissers, Florent Bernard, Johanowen De Craene, Eckhard Boles, Antonio Urrestarazu, 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.
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A C‐terminal di‐leucine motif and nearby sequences are required for NH4+‐induced inactivation and degradation of the general Amino Acid Permease, Gap1p, of Saccharomyces cerevisiae
Molecular Microbiology, 1997Co-Authors: Claudine Hein, Bruno AndreAbstract:: The general Amino Acid Permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the Permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the Permease resistant to NH4(+)-induced inactivation. An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an Amino Acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 Permease against NH4(+)-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the Permease. In contrast, deletion of the last eleven Amino Acids of Gap1p, a region conserved in other Amino Acid Permeases, conferred resistance to NH4+ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive Permeases, namely the arginine (Can1p) and uracil (Fur4p) Permeases.
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a c terminal di leucine motif and nearby sequences are required for nh4 induced inactivation and degradation of the general Amino Acid Permease gap1p of saccharomyces cerevisiae
Molecular Microbiology, 1997Co-Authors: Claudine Hein, Bruno AndreAbstract:: The general Amino Acid Permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the Permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the Permease resistant to NH4(+)-induced inactivation. An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an Amino Acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 Permease against NH4(+)-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the Permease. In contrast, deletion of the last eleven Amino Acids of Gap1p, a region conserved in other Amino Acid Permeases, conferred resistance to NH4+ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive Permeases, namely the arginine (Can1p) and uracil (Fur4p) Permeases.