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

Influencing factors for hiring international students : a case study of MSMEs in the Tampere region

Finland’s population is expected to age rapidly in the coming decades compared to the European Union, the OECD, and other Nordic countries resulting in potential labour shortages and financial pressure on several levels. These repercussions are seen as a threat to Finland’s welfare system and long-term economic growth. Therefore, the country needs international talents to mitigate these challenges. Finland already has a talent pool with 3000 to 4000 international students who graduate each year, however these students still find it very difficult to find an internship during their studies or work after graduation. Hence, the thesis aimed to identify and explore the key factors that influence the hiring decisions for international students with the potential of improving their employability. This study focused on MSMEs in the Tampere region commissioned by the Talent Boost Program. The theoretical framework included an overview of the Tampere region and highlighted the importance of diversity in the workplace. Most of the research on the employability of international students so far has not paid much attention to the theory of planned behaviour. Therefore, attitude, subjective norms, and perceived behavioural control were among the other factors considered by the research to grasp a better understanding of what could influence employers’ hiring decisions. This research is a single exploratory case study in which the data were obtained from both secondary and primary sources. Secondary data were acquired from academic journal articles, official government publications, and other reliable sources. While the primary data were obtained by adopting exploratory sequential mixed methods design, where qualitative and quantitative data were collected by conducting an interview and one questionnaire. The thesis identified and explored the main factors influencing hiring decisions for international students in the Tampere region and confirmed that the career outcomes of international students are influenced not only by their qualifications but also by employers' attitudes, subjective norms, and perceived behavioural control along with other key factors associated with recruitment channels, language proficiency and company resources. Thus, improving the employability of international students requires a long-term action plan that needs cross-level cooperation between all stakeholders in the Tampere region

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

Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe