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. <p>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. <p>In the first part of this work, we report that the Ypq proteins are most probably implicated in the export of basic amino acids from the vacuole to the cytosol. More precisely, Ypq2 and Ypq3 behave like vacuolar arginine and lysine exporters, respectively. Interestingly, the mammalian rPqlc2 protein expressed in yeast reaches the vacuolar membrane and functions as an orthologue of the Ypq proteins. Our results also reveal that the expression of the YPQ3 gene is regulated by the Lys14 transcription factor, responsible for the transcriptional activation of the LYS genes encoding enzymes implicated in the biosynthesis of lysine. We have also noted that, in general, the expression of the expression of the YPQ genes is regulated according to the quality of the nitrogen source available in the extracellular medium, eg. YPQ3 is sensitive to the nitrogen catabolite repression regulatory mechanism. <p>In the last part of this thesis work, we investigated the intracellular trafficking of the Ypq proteins and show that these predominantly reach the vacuolar membrane via the ALP (alkaline phosphatase) pathway due to the presence of a dileucine-based sorting signal in their sequences. Interestingly, a similar mechanism seems responsible for targeting to the yeast vacuole of the mammalian rPqlc2 protein.<p><p><p>Une caractéristique des cellules eucaryotes est leur organisation en compartiment internes délimité par une membrane lipidique, appelé organelles. Ces compartiments intracellulaires présentent une composition lipidique et protéique particulaire conforme à leur identité et fonction. Les lysosomes de cellules de mammifères et la vacuole fongique jouent un rôle clé dans la digestion intracellulaire de macromolécules et de ce fait leurs lumières sont enrichis d’enzymes hydrolytiques nécessaires à cette action. Des disfonctionnements du lysosome peuvent être la conséquence de pathologie chez l’homme, regroupé sous le nom de maladie lysosomale, lié à un à une accumulation de macromolécules non digéré ou un default d’export des produits d’hydrolysé depuis la lumière du lysosome. La cystinose est une maladie autosomale récessive avec une faible fréquence d’incidence (1/200 000) qui regroupe trois formes cliniques :deux formes rénales graves et une forme extra-rénale. Cette maladie est due à une accumulation et cristallisation de cystine dans la lumière du lysosome qui est corrélé à des mutations ponctuelles dans le gène CTNS qui code pour l’exporteur de cystine, la cystinosine. Cette protéine est un membre de la famille LCT (Lysosomal Cystine Transporter) qui possède des représentants chez les cellules animales, végétales et fongiques. Les protéines de la famille possèdent une taille et une topologie prédite similaire (7 segments transmembranaires) et on retrouve aussi au sein de ces protéines deux exemplaires de motifs PQ. Lors de ce travail de thèse nous nous sommes intéressés à trois membres de la famille LCT chez Saccharomyces cerevisiae que nous avons nommé Ypq1, Ypq2 et Ypq3 pour Yeast PQ-loop proteins. Ces protéines n’ayant pas fait l’objet de nombreuses études, nous nous sommes orientés vers une analyse fonctionnelle et transcriptionnelle. De plus, nous avons également étudié les mécanismes et signaux impliqué dans leur adressage vers la vacuole. Finalement, nous avons également inclus dans notre étude un homologue mammalien de ces protéines, rPqlc2. <p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Amyotrophic Lateral Sclerosis (ALS) is a neurological disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have revealed that many ALS-associated genes are involved in autophagy, but the role of this pathway in motor neurons remains poorly understood. Here, we use the SOD1G93A mouse model to investigate the role of autophagy in ALS. We find neuronal subtype-specific regulation of autophagy over the course of disease progression. Vulnerable motor neurons form large GABARAPL1-positive autophagosomes that engulf ubiquitinated cargo recognized by the selective autophagy receptor p62. Other motor neurons and interneurons do not engulf cargo within GABARAPL1-positive autophagosomes and instead accumulate somatodendritic aggregates. To investigate whether motor neuron autophagy is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 is specifically disrupted in motor neurons. Phenotypic analysis of these mice revealed that autophagy is dispensable for motor neuron survival but plays a key role in regulating presynaptic structure and function. By crossing these mice to the SOD1G93A mouse model, we find that autophagy inhibition accelerates early neuromuscular denervation and neurological dysfunction. However, loss of autophagy in motor neurons eventually leads to an extension of lifespan, and this is associated with reduced pathology in interneurons and glial cells. These data suggest that vulnerable motor neurons rely on autophagy to maintain neuromuscular innervation early in disease. However, autophagy eventually acts in a non-cell autonomous manner to promote disease spread and neuroinflammation. Our results reveal counteracting roles for motor neuron autophagy early and late in ALS disease progression.

Autophagy is the process by which cells eliminate its unwanted or dysfunctional components. A major step in autophagy is the formation of autophagosome, the double membrane that engulfs the unwanted cellular components. Dysregulation of autophagy affects neurodegenerative disorders, infectious diseases, cancer, and aging. In yeast, Atg8 protein is considered to play a crucial role in autophagosome maturation. Studies have shown that yeast lacking Atg8 protein form extremely small autophagosomes. Similarly, mammalian cells lacking Atg8 homologues produced “open” autophagosomes. Microtubule-associated protein (MAP) light chain3 (LC3), a human homologue of Atg8 protein is considered to play a major role in autophagosome maturation. However the exact mechanism by which Atg8/LC3 affects the autophagosome maturation is not completely known. A possible mechanism evolving from various studies is the following: Upon binding to the autophagosome, Atg8 family undergoes a conformational transition, which allows it to associate with another membrane-bound Atg8 in a trans-fashion. The proposed goals of this research include testing this hypothesis, identifying the stable conformations of LC3 and LC3-II (membrane bound LC3) and getting insights into the molecular mechanism by which LC3 influence autophagosome maturation. To accomplish this, we are performing Hamiltonian replica exchange molecular dynamics (HREMD) simulations on LC3 and on LC3-II. The most stable conformations of LC3, and LC3-II are identified via clustering analysis. As autophagy modulation is considered as a potential therapeutic target for various diseases, understanding the molecular mechanisms of different stages of autophagy is very important.

La vacuole est un organite qui joue un rôle important dans de nombreux processus de la cellule végétale, et en particulier dans la protection contre les toxiques cellulaires. Parmi ceux-ci, le cadmium est un polluant courant qui affecte les fonctions physiologiques de la plante. La vacuole est connue pour sa capacité à séquestrer les ions métalliques présents dans le cytosol. Toutefois, les acteurs protéiques de cette compartimentation, et notamment les transporteurs, ne sont pas bien connus. Afin de mieux comprendre le rôle de cet organite dans les mécanismes de protection contre le stress métallique, nous avons mis en place une série d'outils d'analyse du protéome vacuolaire, dans le but de réaliser une caractérisation protéomique de référence de cet organite, utile pour l'étude de sa dynamique en conditions de stress. Mon travail a consisté dans un premier temps à mettre au point une méthode de purification de vacuoles à partir de cellules en culture d'Arabidopsis thaliana. La pureté des échantillons obtenus a été confirmée à l'aide de tests biochimiques (western-blots et mesures d'activités enzymatiques) et nous avons pu initier l'analyse par spectrométrie de masse des constituants protéiques de la membrane et de la fraction soluble de la vacuole. Celle-ci a permis d'identifier 689 protéines non-redondantes, dont 110 transporteurs. La localisation in vivo de 5 d'entre elles a été réalisée via l'expression in planta de ces protéines fusionnées à la GFP. Cette première approche protéomique a été complétée d'une étude plus approfondie du protéome vacuolaire, visant à en acquérir des notions quantitatives et organisationnelles. Pour cela, des gels d'électrophorèses bidimensionnelles (IEF / SDS-PAGE) ont été réalisés à partir de la fraction soluble des vacuoles. Les spots résolus ont été quantifiés via le logiciel d'analyse d'images PDQuest et identifiés par spectrométrie de masse. Cette étude a permis de mettre en évidence un certain nombre de protéines majeures de ce compartiment et a porté le nombre total de protéines vacuolaires identifiées par nos travaux à 709. Quelques protéines connues pour être cytosoliques ont toutefois été retrouvées, et une cartographie du protéome cytosolique a également été réalisée afin de la comparer avec celle du protéome soluble de la vacuole et tenter de mieux comprendre l'origine de ces protéines. Cette analyse a été complétée d'une expérience préliminaire de digestion de vacuoles intactes par la protéinase K. Les résultats obtenus suggèrent la présence de protéines à l'intérieur de la vacuole (probablement en cours de dégradation), et d'autres associées à la face externe du tonoplaste probablement de façon spécifique. Enfin, la présence de complexes protéiques a été évaluée à travers la réalisation d'électrophorèses en conditions non dénaturantes qui ont permis de retrouver des complexes connus (ATPase vacuolaire) mais aussi de mettre en évidence plusieurs complexes putatifs de protéines diverses (protéases, glycosidases ...). Un dernier aspect de mon travail a enfin consisté à développer un outil informatique d'exploitation de données d'analyses à haut débit. La confrontation, grâce à cette structure, des résultats de protéomiques vacuolaires et d'autres d'expression génique lors d'un stress cadmium ont permis d'identifier des protéines présentes dans (ou associées à) la vacuole dont le niveau de transcrit est modulé lors d'un stress. Ces analyses croisées ont notamment mis en évidence la protéine DWARF1, qui catalyse une étape de la biosynthèse des brassinostéroïdes, une classe d'hormones. L'étude de l'implication de cette hormone a alors montré que celle-ci est capable de moduler la tolérance au cadmium de plantules, très probablement via des mécanismes qui n'ont encore jamais été identifiés. L'ensemble de ce travail constitue une base pour l'étude ultérieure de la dynamique du protéome vacuolaire. Il propose des méthodes et des outils d'analyse, ainsi qu'une série de données de référence, pour mieux comprendre les processus vacuolaires de la détoxication métallique, et a d'ores et déjà permis de mettre en évidence des aspects nouveaux du fonctionnement de cet organite.

Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.

AP2 complex protein is an essential clathrin adaptor protein during clathrin mediated endocytosis. However, this view has been challenged in simple organisms. To gain insight into this conflict, the role of AP2 in clathrin localization and other clathrin related processes were assessed in Dictyostelium discoideum. In Dictyostelium, deleting function AP2 caused mild phenotypes in clathrin membrane localization, cytokinesis, osmoregulation and cell development. This supported the idea that AP2 have significant roles in multicellular organisms but not in unicellular system. Clathrin mediated processes carries important function not only on the plasma membrane but also on some internal organelles. But clathrin coated vesicles on internal organelles are not as well studied as on the plasma membrane. To understand more of the clathrin coated vesicles on internal organelles, the clathrin coated vesicles on Dictyostelium discoideum contractile vacuole were studied. Contractile vacuole associated clathrin coated vesicles contained clathrin adaptor proteins AP2, AP180, and epsin but not Hip1r. The absence of AP180 or AP2 produced abnormal large vacuoles, but the absence of epsin did not cause any detectable contractile vacuole abnormality. The enlarged contractile vacuoles in AP180 minus cells were caused by excessive homotypic fusion among contractile vacuoles. Using both GST-pull down and immunostaining AP180 was identified as the possible adaptor protein for a contractile vacuole-associated SNARE protein, Vamp7B. Therefore recycling Vamp7B from contractile vacuole by AP180 through clathrin coated vesicles could be an efficient way to prevent excessive homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin coated vesicles on cell internal organelles. / text

The BEACH family proteins are conserved in all eukaryotes and are important for membrane trafficking. Defects in specific BEACH proteins have been linked to severe human disorders. For example, loss of human LYST protein causes the Chediak-Higashi Syndrome (CHS), a lethal disorder that affects lysosomal function. I postulate that different classes of BEACH proteins contribute distinct cellular functions in specific organelles. Based on this functional specificity, I hypothesize that each class of BEACH proteins must localize to their respective organelle where they are known to function. Unfortunately, the localization of most mammalian BEACH proteins is not known and no localization signal has been determined for any BEACH protein. Previous work showed that the Dictyostelium LvsA protein localizes and functions on the contractile vacuole while LvsB localizes and functions on the lysosome. Thus, Dictyostelium is a good model system to understand how BEACH proteins localize to specific organelles. Using a knock-in approach and parasexual techniques, I generated a collection of LvsA truncation mutants tagged with GFP and expressed them in different cell lines. Hence I can test the ability of each mutant protein to localize on contractile vacuoles by fluorescence microscopy. I show here that LvsA requires two regions to localize on the contractile vacuole: the N-terminal 140-457 amino acids and the BEACH. In addition, the expression of the N-terminal 651 amino acids of LvsA causes a dominant negative effect suggesting a possible functional protein-protein interaction within this region. Furthermore, sequence alignment analysis shows that this N-terminal region is only conserved within each class of BEACH family proteins. This finding supports our hypothesis and suggests that diversity within the N-terminal region may be due to the specialized targeting sequences of each class of BEACH proteins. Taken together, these results suggest that the conserved BEACH domain may serve as a general localization sequence while the N-terminal segment is responsible for targeting these proteins to their distinct organelles. This study will facilitate the identification of localization signals in other BEACH proteins which is important to dissect the molecular mechanism of their respective functions. / text

Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.

The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.