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Plasma membrane H+-ATPases are highly conserved in yeast, other fungi and plants. Their main role is to establish the H+ gradient at the plasma membrane (PM) to maintain the cytosolic pH close to neutral values and fuel the uptake of nutrients through secondary active transport systems. In both plants and yeast, PM H+-ATPases have also been described to promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. This for instance occurs in yeast upon H+ influx coupled to amino acid uptake. Fungal and plant PM H+-ATPases are self-inhibited by their carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast PM H+-ATPase (Pma1), the self-inhibitory action of the carboxyterminal tail is neutralized when the adjacent S911 and T912 are phosphorylated, but the kinase(s) and phosphatase(s) controlling their phosphorylation remain unknown.In this thesis work, with the objective of understanding the molecular events taking place at Pma1`s level upon H+ influx and triggering the signaling cascade towards TORC1, we show that the largely redundant Ptk1 and Ptk2 kinases are responsible for phosphorylating S911-T912 at the carboxyterminal tail of Pma1. We also show that the carboxyterminal tail of Pma1 is not essential to initial TORC1 activation induced by H+-coupled amino acid uptake. However, if not properly phosphorylated by Ptk1 and Ptk2 kinases, the carboxyterminal tail of Pma1 can interfere with TORC1 activation. Additionally, we provide evidence that activated TORC1 exerts negative feedback control on Pma1 by reducing phosphorylation of its carboxyterminal tail.Overall, our results shed important new light on phosphoregulation of the yeast Pma1 H+-ATPase and on its interconnections with TORC1., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic, expression and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of putative partners to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. We also show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans. Our results allow to propose a model for the sensing function of Mep2 where pH and calcium are key players., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Chez les eucaryotes, le complexe kinase TORC1 (Target Of Rapamycin Complex 1) joue un rôle central dans le contrôle de la croissance cellulaire. Il intègre de nombreux signaux et agit en modulant l’état de phosphorylation de différents effecteurs, principalement des protéines impliquées dans des processus anaboliques ou cataboliques. Parmi ces signaux, on distingue notamment les acides aminés. Ces derniers agissent sur TORC1 via l’action de protéines de la famille des GTPases Rag, elles-mêmes régulées par des facteurs GEF et GAP. Des études récentes sur des cellules mammifères ont mis en évidence l’existence de senseurs d'acides aminés, capables de moduler l'activité des facteurs GEF et GAP. Chez la levure, ces senseurs restent toutefois inconnus. Chez la levure, TORC1 contrôle la fonction de plusieurs protéines y compris des transporteurs d'acides aminés de la membrane plasmique, comme la perméase générale des acides aminés, Gap1. C’est via sa branche de signalisation Tap2-PP2A/Npr1 que TORC1 contrôle l'ubiquitylation et le trafic intracellulaire de ces transporteurs, et ceci, en modulant l’activité d’adaptateurs de type α-arrestine de l'ubiquitine-ligase Rsp5.Dans ce travail, nous avons combiné des approches de génétique et de biochimie chez la levure afin d’étudier la régulation de TORC1 et son rôle dans l'endocytose en réponse au substrat de Gap1, la perméase générale des acide aminés, et de Can1, la perméase spécifique de l'arginine.Dans la première et la deuxième partie de ce travail, je décris ma contribution à l'étude qui visait à élucider le mécanisme d'ubiquitylation de Gap1 et de Can1 induite par le transport de leurs substrats. Le modèle déduit de ce travail propose que cette régulation négative n'est pas due à l'accumulation intracellulaire des acides aminés transportés, mais à un changement conformationnel des perméases, couplé à la réaction de transport et qui fait apparaitre un état ouvert vers l'intérieur, entraînant ainsi le remodelage de la queue, The Target of Rapamycin Complex 1 (TORC1) plays a pivotal role in controlling cell growth in probably all eukaryotic organisms. It operates by integrating upstream signals like growth factors and nutrients to modulate by phosphorylation multiple downstream effectors, mostly proteins involved in anabolic or catabolic processes. Among the various signals that impinge on TORC1, nitrogen sources, in particular amino acids are primordial input signals modulating TORC1 activity through the conserved Rag family of GTPases. Recent studies in mammals have shed the light on the existence of various sensor systems of internal amino acids that modulate the activity of the GEF and GAP factors acting on the Rag GTPases. Yet, in yeast the amino acid sensing events acting upstream of the Rag GTPase (named Gtr1 and Gtr2) regulators remain poorly known. Yeast TORC1 controls the function of many proteins including several plasma membrane amino acid transporters, e.g. Gap1, the general amino acid permease. It does so via the Tap2-PP2A/Npr1-signaling branch that controls the ubiquitylation and intracellular trafficking of these proteins through regulation of α-arrestin-type adaptors of the ubiquitin-ligase Rsp5. In this work, we combined yeast genetics and biochemical assays to study TORC1 regulation by amino acids and to illustrate the role of TORC1 in substrate-transport mediated endocytosis of Gap1 and of the arginine specific permease, Can1. In the first and second part of this work, I describe my contribution to the study that aimed at elucidating the mechanism of substrate-transport-mediated ubiquitylation and endocytosis of Gap1 and Can1. The model deduced from this work states that this down-regulation is not due to intracellular accumulation of the transported amino acids, but to substrate-transport-induced conformational transition of the transporters to an inward-facing state, resulting in remodeling of their N-terminal cytoplasmic tail and subsequent exposure of a hidden bin, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Proteins of the conserved Mep-Amt-Rh superfamily, including mammalian Rhesus factors,mediate ammonium transport. Ammonium is an important nitrogen source for thebiosynthesis of amino acids for instance but its accumulation is also known as cytotoxic inanimals. Nevertheless, the controlled disposal of ammonium in urine plays a critical role inthe regulation of the acid-base homeostasis. Alteration in ammonium transport via human Rhproteins could have clinical outcomes. In this work, we addressed aspects of structurefunctionanalysis of altered human Rhesus proteins using a heterologous expression systemand further characterized aspects of the patho-physiological roles of Rh proteins usingknockout mice models available in the laboratory.Using a yeast-based expression assay, we characterized human Rh variants resulting from nonsynonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinicalphenotypes. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditarystomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsicbidirectional ammonium transport, suggesting altered ammonium transport as a potentialhallmark of the disease. Moreover, these variants showed trans-dominant negative effects onthe activity of their native HsRhAG counterpart, suggesting altered cooperation of thesubunits in “heteromeric” transport complexes. On the other hand, we revealed that theR202C variant of HsRhCG, the orthologue of mouse Rhcg required for optimal urinaryammonium excretion and blood pH control, shows an impaired inherent ammonium transportactivity. HsRhCGR202C may potentially confer susceptibility to disorders leading to metabolicacidosis for instance. MmRhcg has been shown to be expressed in the male mice epididymal tract, its absenceleading to a more acidic luminal fluid and to a reduced male fertility. Using micemodels, we further investigated the role of Rhcg, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

SUMMARYGap1, the general amino acid permease of Saccharomyces cerevisiae, is a plasma membrane protein which is synthesized and most active under conditions of poor nitrogen supply. Under these conditions, the role of Gap1 is to scavenge external amino acids to be used as nitrogen sources or directly as building blocks for protein synthesis. Gap1 is a member of the Yeast Amino Acid Transporter (YAT) family, a family of amino acid transporters highly conserved in bacteria and fungi. The intracellular trafficking of Gap1 has been the subject of intense investigation as well as the role of lipids (in particular sphingolipids) in its activity and folding. These studies have all been carried out in the cellular context using versatile yeast genetics as exploratory tools. While such in vivo investigations allow to identify physiologically relevant features, they do not provide the details to understand the molecular basis of these phenomena. In order to decipher the molecular features responsible for biological functions, physiological analysis must be combined with biochemical, biophysical and structural studies which typically will be performed on isolated and purified proteins.Our work during this study fits in this trans-disciplinary approach that aims at understanding different properties of the protein: (I) how sphingolipids modulate the activity of Gap1, (II) what part of the protein, and more specifically, what residues, are implicated in its regulation and (III) what are the molecular determinants of the multi-specificity of Gap1.At the beginning of this work, Gap1 had not been previously produced and purified. During this thesis we have identified suitable expression and purification strategies for Gap1 and initiated first characterization studies. In the process, we have developed a new expression system in S. cerevisiae based on the nitrogen catabolite repression. The capacity of this system to express other proteins was successfully tested f, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The cytoplasm of eukaryotic cells contains several membrane-delimited compartments of specific molecular compositions and functions. Among those, the vacuole of fungal cells is often described as an organelle equivalent to the lysosomes of animal cells and the vacuoles of plant cells. These compartments indeed share two similar features: they contain a wide variety of hydrolases and are the most acidic compartments of the cell, which accounts for their key role in the intracellular degradation of macromolecules. In humans, dysfunctions of the lysosomes often give rise to lysosomal related diseases, such as lysosomal storage disorders. These are a class of metabolic disorders caused by the accumulation of non-degraded macromolecules or impaired export of hydrolytic degradation products. Cystinosis is an autosomal recessive disorder (1/200 000 incidence) generally associated with renal dysfunctions. It is caused by the accumulation and crystallization of cystine, the disulfide of cysteine, into the lumen of lysosomes. Cystinosin, the causative gene product of cystinosis, is present at the lysosomal membrane and catalyses the export of cystine from this compartment. The human cystinosin is a member of the Lysosomal Cystine Transporter (LCT) family. LCT proteins are conserved in all eukaryotic species and are defined by the presence of highly conserved PQ-loop motifs. During this thesis work, we have studied three LCT proteins of the yeast Saccharomyces cerevisiae, named Ypq1, Ypq2 and Ypq3 (Yeast PQ-loop proteins 1, 2 and 3). We first showed that these proteins localize to the vacuolar membrane. We next studied the roles of these proteins, the regulation of their genes and the mechanisms and signals implicated in their delivery to the vacuolar membrane. We also contributed to the functional characterization of a mammalian homologue of yeast Ypq proteins, named rPqlc2. In the first part of this work, we report that the Ypq proteins are most probably implicated in, Doctorat en Sciences, info:eu-repo/semantics/nonPublished