Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Signals and mechanisms controlling the ubiquitylation and down-regulation of the yeast general amino acid permease

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.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Stress Conditions Promote Yeast Gap1 Permease Ubiquitylation and Downregulation via the Arrestin-like Bul and Aly Proteins.

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 downregulation 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 here report that Gap1 is also downregulated 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 downregulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this downregulation without undergoing dephosphorylation. Furthermore, they act via 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 downregulated under stress via the Bul and Aly adaptors. While the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, K9 and K16, the Aly proteins promote ubiquitylation of the K16 residue only. This stress-induced pathway of Gap1 downregulation targets other permeases as well, and likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling

Two structurally and functionally distinct mammalian TOR complexes control cell growth and metabolism in physiological and pathological contexts including cancer. Upregulated glutaminolysis is part of the metabolic reprogramming occurring in cancer, providing fuels for growth but also liberating ammonium, a potent neurotoxic waste product. Here, we identify ammonium as a novel dose-dependent signal mediating rapid mTORC2 activation and further regulating mTORC1. We show that ammonium induces rapid RICTOR-dependent phosphorylation of AKT-S473, a process requiring the PI3K pathway and further involving the Src-family kinase YES1, the FAK kinase and the ITGβ1 integrin. Release of calcium from the endoplasmic reticulum store triggers rapid mTORC2 activation, similar to ammonium-induced activation, the latter being conversely prevented by calcium chelation.Moreover, in analogy to growth factors, ammonium triggers the AKT-dependent phosphoinhibition of the TSC complex and of PRAS40, two negative regulators of mTORC1. Consistent with mTORC1 stimulation, ammonium induces the inhibitory phosphorylation of 4EBP1, a negative regulator of protein biogenesis. Ammonium however dually impacts on the phosphorylation of p70S6K1 triggering a transient AKT-independent decrease in the phosphorylation of this second mTORC1 readout. Finally, we reveal ammonium as a dose-dependent stimulator of proliferation. This study underscores an mTORC2 and mTORC1 response to the so-called ammonium waste.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mono-Symptomatic Nocturnal Enuresis in Lebanese Children: Prevalence, Relation with Obesity, and Psychological Effect

Introduction Nocturnal enuresis is involuntary urination while sleeping after a certain age, usually five years, when children should have established bladder control. The prevalence has been found to be up to 20% in five year old children, and it is considered the most common urological childhood complication. Material and Methods This study was conducted on Makassed School children aged 5–18 years. This was a two-step study, the first step was a questionnaire distributed to the children to be answered by their parents. The second step included individually meeting with every child who met the inclusion criteria and his/her parents and physically examining the child. Results 11,440 questionnaires were distributed to school children aged 5–18 years, to be answered by their parents. Of the 7270 parents who responded back, 6620 reported no enuresis, 90 (1.25%) reported only diurnal enuresis, 107 (1.5%) reported diurnal and nocturnal enuresis, and 453 parents reported their child having nocturnal enuresis only. The data collected was analyzed according to age, sex, area, body mass index (BMI), and the PMQOL-SF score. The prevalence of mono-symptomatic nocturnal enuresis (MNE) in Lebanon was found to be 5.3%. The results showed that the prevalence of MNE is inversely proportional to age. The prevalence of male to female ratio was 1.4:1. As for the prevalence according to different geographic areas, the results have shown that the North had the majority of cases with 7.6% prevalence. Results showed that 82.4% of children had a score more than 50, and only 28% of parents had a score above 50. Discussion The prevalence of nocturnal enuresis in Lebanon is lower than that in neighboring countries such as Turkey 8 and Saudi Arabia, 9 but higher than that in Italy 10 and Hong Kong. Our study has managed to show the same results, with a peak in incidence at seven years then dropping back to 0% at the age of 16. Our study has shown a male to female predominance but the male to female ratio was 1.4:1, a value lower than that described in earlier studies. Our study has shown that more than 80% of children were psychologically affected whereas only less than 30% of parents were affected. Conclusion To our knowledge, this was the first study in Lebanon conducted to determine the prevalence of MNE. The relatively low prevalence rate found may be because of differences in genetic predisposition, psychosocial or environmental conditions, and traditional and cultural backgrounds. No relation was found between obesity and nocturnal enuresis. The psychological impact on children is significant but that on the parents is minimal

Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae.

Decades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32.

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.SCOPUS: ar.jinfo:eu-repo/semantics/publishe