Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.

No description available.

No description available.

Vitamin E wird immer noch als das wichtigste lipophile Antioxidanz in biologischen Membranen betrachtet. In den letzten Jahren hat sich jedoch der Schwerpunkt der Vitamin E-Forschung hin zu den nicht-antioxidativen Funktionen verlagert. Besonderes Interesse gilt dabei dem α-Tocopherol, der häufigsten Vitamin E-Form im Gewebe von Säugetieren, und seiner Rolle bei der Regulation der Genexpression. Das Ziel dieser Dissertation war die Untersuchung der genregulatorischen Funktionen von α-Tocoperol und die Identifizierung α-Tocopherol-sensitiver Gene in vivo. Zu diesem Zweck wurden Mäuse mit verschiedenen Mengen α-Tocopherol gefüttert. Die Analyse der hepatischen Genexpression mit Hilfe von DNA-Microarrays identifizierte 387 α-Tocopherol-sensitive Gene. Funktionelle Clusteranalysen der differentiell exprimierten Gene zeigten einen Einfluss von α-Tocooherol auf zelluläre Transportprozesse. Besonders solche Gene, die an vesikulären Transportvorgängen beteiligt sind, wurden größtenteils durch α-Tocopherol hochreguliert. Für Syntaxin 1C, Vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and Syntaxin binding protein 1 konnte eine erhöhte Expression mittels real time PCR bestätigt werden. Ein funktioneller Einfluss von α-Tocopherol auf vesikuläre Transportprozesse konnte mit Hilfe des in vitro β-Hexosaminidase Assays in der sekretorischen Mastzelllinie RBL-2H3 gezeigt werden. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer konzentrationsabhängigen Erhöhung der PMA/Ionomycin-stimulierten Sekretion der β-Hexosaminidase. Eine erhöhte Expression ausgewählter Gene, die an der Degranulation beteiligt sind, konnte nicht beobachtet werden. Damit schien ein direkter genregulatorischer Effekt von α-Tocopherol eher unwahrscheinlich. Da eine erhöhte Sekretion auch mit β-Tocopherol aber nicht mit Trolox, einem hydrophilen Vitamin E-Analogon, gefunden wurde, wurde vermutet, dass α-Tocopherol die Degranulation möglicherweise durch seine membranständige Lokalisation beeinflussen könnte. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer veränderten Verteilung des Gangliosids GM1, einem Lipid raft Marker. Es wird angenommen, dass diese Membranmikrodomänen als Plattformen für Signaltransduktionsvorgänge fungieren. Ein möglicher Einfluss von Vitamin E auf die Rekrutierung/Translokation von Signalproteinen in Membranmikrodomänen könnte die beobachteten Effekte erklären. Eine Rolle von α-Tocopherol im vesikulären Transport könnte nicht nur seine eigene Absorption und seinen Transport beeinflussen, sondern auch eine Erklärung für die bei schwerer Vitamin E-Defizienz auftretenden neuronalen Dysfunktionen bieten. Im zweiten Teil der Arbeit wurde die α-Tocopheroltransferprotein (Ttpa) Knockout-Maus als genetisches Modell für Vitamin E-Defizienz verwendet, um den Effekt von Ttpa auf die Genexpression und die Gewebeverteilung von α-Tocopherol zu analysieren. Ttpa ist ein cytosolisches Protein, das für die selektive Retention von α-Tocopherol in der Leber verantwortlich ist. Die Ttpa-Defizienz resultierte in sehr geringen α-Tocopherol-Konzentrationen im Plasma und den extrahepatischen Geweben. Die Analyse der α-Tocopherol-Gehalte im Gehirn wies auf eine Rolle von Ttpa bei der α-Tocopherol-Aufnahme ins Gehirn hin. / Vitamin E is still considered the most important lipid-soluble antioxidant within biological membranes. However, in the last years the non-antioxidant functions of vitamin E have become the focus of vitamin E research. From the eight members of the vitamin E family, specific emphasis is given to α-tocopherol, the most abundant vitamin E form in mammalian tissues, and its role in the regulation of gene expression. The aim of this thesis was the analysis of the gene regulatory functions of α-tocopherol and the identification of α-tocopherol sensitive genes in vivo. For this purpose mice were fed diets differing in α-tocopherol content. The analysis of hepatic gene expression using DNA microarrays identified 387 α-tocopherol-sensitive genes. Functional cluster analyses of these differentially expressed genes demonstrated an influence of α-tocopherol on cellular transport processes. Especially the expression of genes involved in vesicular trafficking was largely upregulated by α-tocopherol. Upregulation of syntaxin 1C, vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and syntaxin binding protein 1 was verified by real time PCR. A role of α-tocopherol in exocytosis was shown by the in vitro β-hexosaminidase release assay in the secretory mast cell line RBL-2H3. Incubation with α-tocopherol resulted in a concentration dependent increase of PMA/ionomycin-stimulated secretion of β-hexosaminidase. Induction of selected genes involved in degranulation was not observed at any time point. Thus, a direct gene-regulatory effect of α-tocopherol seemed rather unlikely. Since increased secretion was also observed with ß-tocopherol but not with trolox, a water-soluble analog of vitamin E, it was hypothesized that α-tocopherol might affect degranulation through its localization at the plasma membrane. Incubation of cells with α-tocopherol changed the distribution of the gangliosid GM1, a Lipid raft marker. These membrane microdomains are assumed to function as signaling platforms. An possible influence of vitamin E on the recruitment/translocation of signaling proteins into membrane microdomains could explain the observed effects. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency. In the second part of this dissertation the α-tocopherol transfer protein (Ttpa) knockout-mouse as a model of genetic vitamin E deficiency was used to analyze the effect of Ttpa gene expression and tissue distribution of α-tocopherol. Ttpa is a cytosolic protein, which is responsible for the selective retention of α-tocopherol in the liver. Its deficiency resulted in very low α-tocopherol concentrations in plasma and extrahepatic tissues. Analysis of α-tocopherol contents in brain indicated a role for Ttpa in the uptake of α-tocopherol into the brain.

Les cellules eukaryotes sont compartimentées par des structures intracellulaires nommées organites. On peut citer le réticulum endoplasmique, l'appareil de Golgi, le réseau endosomal et lyzosomal. Ces structures délimitées par des membranes cellulaires sont hautement dynamiques, structures dont les composants s'échangent sans cesse entre les différents compartiments. Malgré cette dynamique, les structures qui composent les réseaux d'organites sont très stables et robustes, de sorte que l'on peut décrire un état stationnaire pour ces systèmes hors équilibre et auto-organisés. Bien qu'ils soient robustes en conditions physiologiques, ces compartiments peuvent subir des modification de structures en condition pathologiques ou sous l'effet de traitements pharmacologiques. L'auto-organisation de systèmes à l'équilibre et relativement bien compris par le biais de diagrammes de phases, où l'on peut représenter lesdites phases en fonctions de paramètres physiques, tels que la concentration, ou les interaction entre les différents composants. La situation est bien moins prédictible pour des systèmes hors équilibre. C'est là donc une question scientifique intéressante que de comprendre les mécanismes contraignant l'organisation intracellulaire, où transports actifs et modification biochimiques des composant, tout deux consommant de l'énergie, sont en compétition avec des phénomènes passifs telle que la diffusion. Nous étudions, aussi bien numériquement qu'analytiquement, des modèles d'auto-organisation et de transport, dans des systèmes où un nombre réduit de composants s'organisent par le biais de réaction stochastiques, en des structures de grandes tailles. La question principale que nous posons est de comprendre comment les dynamiques d'échanges entre compartiments (par le biais de vésiculations et de fusion) jouent de concert avec les cinétiques de maturation des composants d'organites, permettent la mise en place d'un réseau robuste. A cette fin, nous nous focalisons sur un organite type, multi-compartiments, doté d'une dynamique riche de transport et de maturation de ses composants : l'appareil de Golgi. Nous décrivons et analysons l'état stationnaire de ces systèmes, en des termes de tailles et de pureté des compartiments le composant - sont ils gros ou petit, triés dans leur composition ou mixés. De cet état stationnaire émerge spontanément un transport de vésicules entre les compartiments, dont la directionnalité est intimement liée à l'état stationnaire. Ce transport est antérograde dans les régimes triés, rétrograde dans les régimes mixés. Des interactions locales, entre les compartiments et ce qu'ils renferment (protéines dont le nom générique est cargo), suffisent à biaiser ces dynamiques de transport. Cela impacte à la fois le temps de résidence des cargos, mais aussi leur localisation dans le système. La capacité de cet organite à trier ces cargos dépend cependant grandement de l'état stationnaire précédemment décrit. / Eukaryotic cells are highly compartmentalized into intracellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, endosomes and lysosomes. These are dynamical structures bounded by lipid membranes, within which components undergo biochemical modification by enzymes, and between which components are constantly being exchanged. Despite their highly dynamical nature, their spatial organization is fairly well conserved over time, so that they could be seen as stationary states of a highly non-equilibrium, and multi-component system. On the other hand, this organization has been observed to be totally disorganized in pathologies or drug treatments. Self-organization in equilibrium systems is fairly well understood by means of phase diagrams where the occurrence of different phases (dispersed, condensed, phase separated) depends on physical parameters (concentrations, interaction energy between components). The situation is much less clear for non-equilibrium systems. It is therefore an exciting challenge to reach a quantitative understanding of the mechanisms dictating the intra-cellular organization, where active transport and biochemical modification by energy-consuming enzymes compete with purely passive phenomena such as diffusion. We design and study, both analytically and numerically, simple models of self-organization and transport in systems where a limited number of components may self-organize into larger structures by means of stochastic reactions. Our main fundamental question is to determine how the interplay between the dynamics of inter-organelle exchange (by means of vesicle secretion, transport and fusion) and the kinetics of biochemical maturation within organelles may yield a precise and robust organelle network. To this end we focus on one "stereotype" organelle, that is already multi-compartments and with a very rich dynamics of vesiculation, fusion and maturation: the Golgi Apparatus. We describe and understand the steady-state organization of such systems, in term of compartments' size and purity - how big and well sorted are the different compartments. From this steady-state, a vesicular transport spontaneously emerges, whose directionality is linked to the steady-state organization. It is anterograde in a pure regime, and retrograde in a mixed configuration. Local interaction between components being transported, and membranes are sufficient to bias those transport. This both change the kinetics of transport in the system, and thus their location in the compartments. How efficient the system is in sorting these elements, strongly relies on the steady-state organization and the vesicular transport.

No description available.

Os fungos filamentosos passam por um crescimento polarizado, desde a germinação ao alongamento das hifas, até formar um complexo micélio. A região apical do crescimento polarizado do fungo apresenta dois tipos diferentes de vesículas, entre elas, as microvesículas. As ADP-ribosylation factors (ARFs), são proteínas monoméricas ligadoras de GTP e pertence ao grupo de proteínas da superfamília Ras. Essas proteínas são divididas em cinco famílias: ARF, RAB, RAN, RAS e RHO que formam um conjunto de sub-sistemas que são responsáveis, entre outras funções, pela regulação do transporte de vesículas no interior da célula fúngica, entre outras funções, como transduções de sinais e regulação do tráfego vesicular na região de crescimento apical, o spitzenkörper. São proteínas de ancoramento e de marcação de vesículas, envolvidas no tráfego, catálise e fusão por meio de sinalização de membrana-alvo para as vesículas de transporte transmembrana. As ARF são importantes para o crescimento das hifas, além de participar da montagem de vesículas por meio de endocitoses, do transporte destas vesículas entre as organelas e na exocitose. Adicionalmente, as ARFs sofrem o processo de N-miristoilação, uma irreversível lipidação proteica em que o miristato do miristoil CoA é covalentemente ligado a uma glicina secundária da proteína alvo, aumentando a sua hidrofobicidade. Além desta regulação, as ARFs são moduladas pela ação das ARF-GAP (GTPase Activating Protein) e ARF-GEFs (Guanine nucleotide Exchange Factor). Neste trabalho foi proposta a deleção de três ARFs preditivamente miristoiladas (arfA, arfB and arlA), além de dupla-deleção com ?gcsA (ARF-GAP) e a caracterização genotípica e fenotípica das ADP ribosylation fator no fungo filamentoso patogênico Aspergillus fumigatus. Como caracterização das linhagens deletadas, notou-se que arfA demonstra ser essencial para A. fumigatus, enquanto que o fungo foi capaz de se desenvolver na ausência de arfB, arlA e duplo mutantes com ?gcsA. Porém, de forma alternada nas linhagens mutantes, houve redução do diâmetro da colônia, desestruturação de conidióforos, polarização dicotômica e redução de corpos lipídicos na região de crescimento apical. Além das alterações da parede celular que implicou em altações na carga de superfície, formação de biofilme e virulência. Testes de sensibilidades, bem como as análises de níveis de expressão gênica frente a a compostos danosos a eucariotos e antifúngicos evidenciaram que as ARFs e GcsA estão envolvidas em reparos a danos frente a diferentes alvos citoplasmáticos. Ainda, a localização das ARFs fusionadas com GFP (Green Flourescence Protein) em A. fumigatus evidenciou que ArfB está nas regiões apicais das hifas e conidióforos, enquanto ArlA está distribuído em todo citoplasma. Portanto as ARFs em A. fumigatus estão envolvidas nos processos básicos do fungo, como: o crescimento, a virulência e a reprodução / The filamentous fungi undergo polarized growth, from germination to hyphae elongation, to form a mycelial complex. The apical region of the polarized growth of the fungus presents two different types of vesicles, among them, the microvesicles. ADP-ribosylation factors (ARFs) are monomeric GTP-binding proteins and belong to a group of superfamily Ras proteins. These proteins are divided into five families: ARF, RAB, RAN, RAS and RHO that form a set of subsystems that are responsible, over others things, for the regulation of vesicle transport within the fungal cell, among other functions, such as signal transduction and regulation of the vesicular traffic in the apical growth region, the Spitzenkörper. They are anchoring and vesicle marking proteins involved in trafficking, catalysis and fusion by means of target membrane signaling to the transmembrane transport vesicles. ARFs are important for the growth of hyphae, besides participating in vesicle assembly through endocytosis, the transport of these vesicles between the organelles and exocytosis. In addition, the ARFs undergo the N-myristoylation process, an irreversible protein lipidation in which the myristoyl CoA myristate is covalently linked to a secondary glycine of the target protein, increasing its hydrophobicity. In addition to this regulation, the Arfs are modulated by the action of Arf-GAP (GTPase Activating Protein) and ARF-GEFs (Guanine nucleotide Exchange Factor). In this work, the deletion of three myristoylated ARFs (arfA, arfB and arlA), as well as double-deletion with ?gcsA (ARF-GAP) and phenotypic and genotypic characterization of ADP ribosylation fator in the pathogenic fungus Aspergillus fumigatus was proposed. As a characterization of the deleted strains, arfA shown to be essential for A. fumigatus, whereas the fungus was able to develop in the absence of arfB, arlA and double mutants with ?gcsA. However, in the mutant strains, there was a decrease in colony diameter, deconjugation of conidiophores, dichotomous polarization and reduction of lipid bodies in the apical growth region. In addition, cell wall changes were registered that implied in surface charge elevations, biofilm formation and virulence. In tests of sensitivities, as well as the analysis of levels of gene expression against compounds harmful to eukaryotes and antifungals showed that ARFs and GcsA (Arf-GAP) are involved in damage repair against different cytoplasmic targets. Furthermore, the location of the GFP-fused GFPs (Green Flourescence Protein) in A. fumigatus evidenced that ArfB is in the apical regions of the hyphae and conidiophores, while ArlA is diffuse in every cytoplasm. Therefore, the ARFs in A. fumigatus are involved in the basic processes of the fungus, such as growth, virulence and reproduction

No description available.

Récemment, le concept monofonctionnel de la protéine Tau en tant que protéine stabilisatrice des microtubules a été remis en cause. Ces nouvelles fonctions sont liées à de nouvelles localisations comme le noyau, la membrane, la synapse ou encore les vésicules. La localisation extracellulaire est particulièrement intéressante car elle pourrait intervenir dans la sécrétion de Tau et expliquer l’évolution hiérarchisée de certaines tauopathies sporadiques dont fait partie la maladie d’Alzheimer. La pathologie Tau peut être induite chez l’animal par injection intracrânienne d’espèces pathologiques et semble se transmettre d’un neurone à un autre et d’une région à une autre. Ce phénomène suit des voies neuroanatomiques et suggère une propagation active des assemblages toxiques des protéines Tau. Des études in vitro ont mis en évidence que les protéines Tau sont capables de se déplacer d’une cellule à une autre propageant ainsi la pathologie par un mécanisme de recrutement des espèces saines. L’existence d’une progression hiérarchisée de la pathologie Tau combinée à sa localisation extracellulaire permet de formuler une nouvelle hypothèse. La protéine Tau serait une protéine de type prion et se comporterait comme telle pour propager la pathologie.Cette caractéristique implique l’existence de mécanismes cellulaires de transports actifs pour transférer les protéines pathologiques. Plusieurs travaux ont montré que la protéine Tau est libérée dans le milieu extracellulaire ou enfermée dans des vésicules extracellulaires lors de son transport entre les cellules. Parallèlement aux mécanismes de sécrétion/capture, des ponts membranaires établissant un contact direct entre deux cellules pourraient être impliquer dans la propagation de Tau. Les TNTs constituent une piste sérieuse de part leur rôle déjà établi dans le transfert de pathogènes et de protéines mal repliées impliqués dans différentes maladies neurodégénératives. Notre objectif a donc été d’étudier l’implication de ces structures dans le transfert interneuronal des assemblages de protéines Tau.Dans ce travail de thèse, nous démontrons que les espèces pathologiques de Tau empruntent les TNTs pour leur transfert interneuronal. Nous apportons les preuves, par vidéo-microscopie, de l’existence d’un transfert de protéines Tau pathologiques d’un neurone primaire à un neurone secondaire et donc d’une implication potentielle des TNTs dans la propagation de la pathologie Tau et la transmission de la maladie. Fait remarquable, la présence des fibres Tau au niveau extracellulaire active la formation des TNTs et facilite leur transfert. Ce résultat place les TNTs au coeur du processus pathologique de la propagation et de son cycle infernal (transfert de Tau dans les cellules naïves par les TNTs – seeding - mort neuronal - libération de Tau dans le milieu extracellulaire - augmentation du nombre des TNTs…). Nous avons aussi apporté une caractérisation des TNTs dans les neurones primaires. Ce résultat est d’autant plus important qu’il est difficile d’identifier des TNTs dans les neurones et c’est dans ce contexte que nous avons réalisé une découverte étonnante, la protéine Tau endogène est présente de manière physiologique dans les TNTs de neurones primaires. Ces résultats révèlent, et pour la première fois, que la protéine Tau, comme l’actine, peut être considérée comme une composante constitutive des TNTs dans les neurones. Elle pourrait ainsi être utilisée comme un marqueur des TNTs. Ces résultats mettent également en lumière une nouvelle fonction de Tau appuyant une fois de plus le caractère multifonctionnel de cette protéine [...] / Over the past few years, the monofunctional concept of Tau protein as a microtubule-associated stabilizing protein has been challenged. These new functions are linked to new localizations: nucleus, membrane, synapse or vesicles. The extracellular localization is particularly interesting as it could play a role in the secretion of Tau and explain the hierarchical evolution of some sporadic tauopathies such as Alzheimer's disease. The Tau pathology can be induced in animals by intracranial injection of pathological species and seems to be transferred from one neuron to another and from one region to another. This phenomenon follows neuroanatomic pathways and suggests an active propagation of the toxic assemblies of Tau proteins. In vitro studies have shown that proteins are able to move from one cell to another and induce the same abnormal conformation of endogenous Tau proteins initiating a self-amplifying cascade. The existence of a hierarchical progression of the Tau pathology combined with its extracellular localization enables to express a new hypothesis. The Tau protein would be a prion-like protein and would behave like that to propagate the pathology.This characteristic implies the existence of cellular active transport mechanisms to transfer pathological proteins. Several studies have shown that the Tau protein, during transport between cells, is released in the extracellular medium or enclosed in extracellular vesicles. Simultaneously with secretion / capture mechanisms, membrane bridges, establishing direct contact between two cells, could be involved in Tau propagation. TNTs are a serious candidate with their already established role in the transfer of pathogens and misfolding proteins involved in various neurodegenerative diseases. Thus, our objective was to study the involvement of these structures in the interneuronal transfer of Tau protein assemblies.In this thesis, we demonstrate that Tau pathological species use TNTs for their interneuronal transfer. We bring evidences, by videomicroscopy, that pathological Tau proteins are transferred from a primary to a secondary neuron and that TNTs could be involved in the spreading of Tau pathology and the disease transmission. Furthermore, the presence of extracellular Tau fibers can activate the formation of TNTs and facilitate their transfer. This result places TNTs in a central place for propagation pathological process and its vicious cycle (transfer of Tau in naive cells by TNTs - seeding - neuronal death – release of Tau in the extracellular environment - increase in the number of TNTs…). We also made a characterization of the TNTs in primary neurons. This result is really important as it is really complex to identify TNTs in neurons. And in this context, we made a surprising discovery: the endogenous Tau protein is physiologically present in TNTs in primary neurons. These results reveal, for the first time, that the Tau protein, like actin, can be considered as a constitutive component of TNTs in neurons. Thus, it could be used as a marker for TNTs. All these results also highlight a new Tau function and reinforce the multifunctional characteristic of this protein.To confirm the importance of this new pathway in the pathological process, further studies should be considered by analyzing if the transfer of pathological Tau species induces a pathological phenotype in the recipient cell and by looking for the cellular mechanisms involved in the transfer of toxic Tau assemblies by TNTs. In vivo studies on integrated systems such as Caenorhabditis elegans would confirm the involvement of these dynamic structures in the pathological process and identify a new therapeutic target.

Vpu has been shown to possess two distinct roles in the pathogenesis of HIV. First, Vpu has been shown to down-regulate the expression of CD4 molecules at the plasma membrane of infected cells by targeting CD4 molecules for degradation in the endoplasmic reticulum. Second, Vpu promotes viral egress in specific cell lines termed non-permissive cells by mechanism that remain relatively unclear. Therefore, experiments were conducted in order to identify cellular factors involved in the Vpu-dependent phenotype. Using full-length Vpu as bait in yeast two-hybrid experiments, several candidate cellular factors were identified. One protein, SNAPIN, was identified as a cellular factor putatively involved in the Vpu-dependent phenotype. Further experiments determined that not only do SNAPIN and Vpu interact, but that Vpu also leads to the degradation of SNAPIN by both proteasomal and lysosomal degradation pathways. Over-expression of SNAPIN in cell lines that do not normally require Vpu expression for viral production resulted in a Vpu-dependent phenotype. While over-expression of SNAPIN in otherwise permissive cell lines significantly reduced Vpu-deficient virus production, wild type levels remained relatively constant. Importantly, no defective viral structural protein production was observed; however, intracellular p24/p55 did not accumulate suggesting that in SNAPIN expressing cells, Gag is also targeted for degradation. In addition, the reduction of SNAPIN expression in non-permissive cell lines significantly increased viral titers in supernatants. Of particular interest, even in cells expressing Bst-2 (a previously identified cellular factor involved in the Vpu-phenotype), siRNA mediated knockdown of SNAPIN led to increased viral titers. In addition, the co-transfection of siRNAs targeting both SNAPIN and Bst-2 resulted in an additive effect, in which Vpu-deficient viral titers were nearly equivalent to wild-type titers. Surprisingly, siRNA-mediated knockdown of SNAPIN in Jurkat cells was sufficient to overcome any restriction in viral egress imposed by the deletion of Vpu. Conversely, siRNA targeting Bst-2 had little or no effect on viral titers in Jurkat cells regardless of whether it was transfected alone or in combination with siRNAs targeting SNAPIN. These experiments provide evidence of an alternate cellular restriction mechanism involved in viral egress that is countered by the HIV-1 accessory protein, Vpu. In addition, this research may provide further insight into the complex cellular networks involved in the trafficking of Gag through cellular endosomal pathways.