Search

Protein secretion is an essential cellular process that permits cell growth, movement and communication. Traffic of proteins within the eukaryotic secretory pathway is mediated by transport intermediates that bud from one compartment, populated with appropriate cargo proteins, and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human PCSK9 is a major determinant of cholesterol metabolism, whose secretion is mediated by a specific cargo adaptor of the ER export machinery, SEC24A. We map a series of protein-protein interactions between PCSK9, its ER export receptor, SURF4, and SEC24A, that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-PBA, a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments. ispartof: JOURNAL OF CELL BIOLOGY vol:220 issue:9 ispartof: location:United States status: published

TANGO1 (transport and Golgi organization‐1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER‐Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild‐to‐moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near‐total bone loss and phenocopies the situation of Mia3 −/− mice. Whole‐exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4‐base pair (4‐bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1‐related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe‐to‐lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

As a major anabolic pathway, the secretory pathway needs to adapt to the demands of the surrounding environment and responds to different exogenous signals and stimuli. In this context, the transport in the early secretory pathway from the endoplasmic reticulum (ER) to the Golgi apparatus appears particularly regulated. For instance, protein export from the ER is critically stimulated by growth factors. Conversely, nutrient starvation also modulates functions of the early secretory pathway in multiple ways. In this review, we focus on amino-acid starvation and how the function of the early secretory pathway is redirected to fuel autophagy, how the ER exit sites are remodeled into novel cytoprotective stress assemblies, and how secretion is modulated in vivo in starving organisms. With the increasingly exciting knowledge on mechanistic target of rapamycin complex 1 (mTORC1), the major nutrient sensor, it is also a good moment to establish how the modulation of the secretory pathway by amino-acid restriction intersects with this major signaling hub.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates glucose, lipid, and energy homeostasis. While gene expression of FGF21 is regulated by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha in the fasted state, little is known about the regulation of trafficking and secretion of FGF21. We show that mice with a mutation in the Yip1 domain family, member 6 gene (Klein-Zschocher [KLZ]; Yipf6 KLZ/Y ) on a high-fat diet (HFD) have higher plasma levels of FGF21 than mice that do not carry this mutation (controls) and hepatocytes from Yipf6 KLZ/Y mice secrete more FGF21 than hepatocytes from wild-type mice. Consequently, Yipf6 KLZ/Y mice are resistant to HFD-induced features of the metabolic syndrome and have increased lipolysis, energy expenditure, and thermogenesis, with an increase in core body temperature. Yipf6 KLZ/Y mice with hepatocyte-specific deletion of FGF21 were no longer protected from diet-induced obesity. We show that YIPF6 binds FGF21 in the endoplasmic reticulum to limit its secretion and specifies packaging of FGF21 into coat protein complex II (COPII) vesicles during development of obesity in mice. Levels of YIPF6 protein in human liver correlate with hepatic steatosis and correlate inversely with levels of FGF21 in serum from patients with nonalcoholic fatty liver disease (NAFLD). YIPF6 is therefore a newly identified regulator of FGF21 secretion during development of obesity and could be a target for treatment of obesity and NAFLD.

SREBPs are master regulators of lipid homeostasis and undergo sterol‐regulated export from ER to Golgi apparatus for processing and activation via COPII‐coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet‐associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP‐Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet‐induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.

van Leeuwen et al. discuss how the cellular secretory pathway adapts and contributes to stress-induced pathways during nutrient starvation., As a major anabolic pathway, the secretory pathway needs to adapt to the demands of the surrounding environment and responds to different exogenous signals and stimuli. In this context, the transport in the early secretory pathway from the endoplasmic reticulum (ER) to the Golgi apparatus appears particularly regulated. For instance, protein export from the ER is critically stimulated by growth factors. Conversely, nutrient starvation also modulates functions of the early secretory pathway in multiple ways. In this review, we focus on amino-acid starvation and how the function of the early secretory pathway is redirected to fuel autophagy, how the ER exit sites are remodeled into novel cytoprotective stress assemblies, and how secretion is modulated in vivo in starving organisms. With the increasingly exciting knowledge on mechanistic target of rapamycin complex 1 (mTORC1), the major nutrient sensor, it is also a good moment to establish how the modulation of the secretory pathway by amino-acid restriction intersects with this major signaling hub.

The upregulation of autophagosome formation in response to nutrient deprivation requires significant intracellular membrane rearrangements that are poorly understood. Recent findings have implicated COPII-coated vesicles, well known as ER-Golgi cargo transport carriers, as key players in macroautophagy. The role of COPII vesicles in macroautophagy and how they interact with autophagy-related (Atg) proteins was unknown. In our recent report, we show that during nutrient deprivation, phosphorylation of the membrane-distal surface of the COPII coat subunit Sec24 facilitates the interaction of Sec24 with the Atg machinery (specifically, Atg9) to regulate the abundance of autophagosomes during starvation. Phosphorylation of Sec24 is specifically required for macroautophagy, but not ER-Golgi transport. These findings begin to unravel the unique function of COPII vesicles during starvation-induced macroautophagy.

Efficiency and fidelity of protein secretion are achieved thanks to the presence of different steps, located sequentially in time and space along the secretory compartment, controlling protein folding and maturation. After entering into the endoplasmic reticulum (ER), secretory proteins attain their native structure thanks to specific chaperones and enzymes. Only correctly folded molecules are allowed by quality control (QC) mechanisms to leave the ER and proceed to downstream compartments. Proteins that cannot fold properly are instead retained in the ER to be finally destined to proteasomal degradation. Exiting from the ER requires, in most cases, the use of coated vesicles, departing at the ER exit sites, which will fuse with the Golgi compartment, thus releasing their cargoes. Protein accumulation in the ER can be caused by a too stringent QC or by ineffective transport: these situations could be deleterious for the organism, due to the loss of the secreted protein, and to the cell itself, because of abnormal increase of protein concentration in the ER. In both cases, diseases can arise. In this review, we will describe the pathophysiology of protein folding and transport between the ER and the Golgi compartment.

Endoplasmic Reticulum (ER)-derived COPII coated vesicles constitutively transport secretory cargo to the Golgi. However, during starvation-induced stress, COPII vesicles have been implicated as a membrane source for autophagosomes, distinct organelles that engulf cellular components for degradation by macroautophagy (hereafter called autophagy). How cells regulate core trafficking machinery to fulfill dramatically different cellular roles in response to environmental cues is unknown. Here we show that phosphorylation of conserved amino acids on the membrane-distal surface of the Saccharomyces cerevisiae COPII cargo adaptor, Sec24, reprograms COPII vesicles for autophagy. We also show casein kinase 1 (Hrr25) is a key kinase that phosphorylates this regulatory surface. During autophagy, Sec24 phosphorylation regulates autophagosome number and its interaction with the C-terminus of Atg9, a component of the autophagy machinery required for autophagosome initiation. We propose that the acute need to produce autophagosomes during starvation drives the interaction of Sec24 with Atg9 to increase autophagosome abundance. DOI: http://dx.doi.org/10.7554/eLife.21167.001, eLife digest When cells experience stressful conditions, such as a shortage of nutrients, they can digest their own material via a ‘self-eating’ process called autophagy and then recycle the products for further use. When autophagy is triggered, a new membrane structure called the autophagosome forms within the cell as it engulfs the material that is to be digested. The autophagosome delivers these materials to a compartment where they are broken down into smaller parts and the resulting raw materials are reused as needed. The membranes that make up the autophagosome are derived from other membranes within the cell. These include small membrane-bound compartments called vesicles, which carry proteins from one part of the cell to another, or to the outside of the cell. COPII vesicles, for example, carry out the first transport step in the pathway that leads out of the cell – the so-called secretory pathway. Recently it was found that, when cells are starving, COPII vesicles can be diverted to the autophagy pathway and provide a source of membrane to build the autophagosome. However, it was not understood how the membrane of a COPII vesicle is reprogrammed so that it can interact with the cellular machinery that builds autophagosomes. Using genetic and biochemical methods, Davis et al. have now teased apart the distinct roles of COPII vesicles in autophagy and the secretory pathway in budding yeast. The results show that a protein called Sec24, a component of the coat on the vesicles, interacts with another protein called Atg9, which is needed for the first steps of autophagosome formation. Davis et al. observed that Sec24 could be modified by the attachment of phosphate groups at a distinct site on the surface of Sec24. This modification promotes Sec24 to interact with Atg9 and increases the number of autophagosomes that form when cells are starving. Davis et al. also found that the enzyme casein kinase 1 is one of the enzymes responsible for attaching phosphate groups to Sec24. Following on from this work, it will be important to test whether modification of vesicle coat proteins is a widespread mechanism for reprogramming membranes for different uses in other situations as well. DOI: http://dx.doi.org/10.7554/eLife.21167.002

The aim of this bachelor thesis is to summarize all published findings about the role of the Erv14 protein in the cells of Saccharomyces cerevisiae. The Erv14 protein works as a cargo receptor in the endoplasmic reticulum (ER), allowing an incorporation of selected trans-membrane proteins to the COPII vesicles, by which their transport to the Golgi apparatus is allowed. Many proteins were identified in the cells of S. cerevisiae, their transport from the ER depends on the presence of the Erv14 protein. In cells lacking Erv14, these proteins are retained in the ER and are not able to fulfil their functions. One of those proteins is for example Axl2, necessary for axial budding, Sma2, which ensures the standard formation of the prospore membrane during sporulation, the catalytic subunit of the chitin synthase III, Chs3, important for chitin synthesis, and plasma membrane Na+ , K+ /H+ antiporter Nha1 participating in the maintenance of cell alkali-metal-cation homeostasis. The importance of the Erv14 protein's function is proven by the fact that its homologs were also found in other organisms, such as animals, including human, as well as in plants. Keywords: Erv14, cargo receptor, COPII vesicles, transport, Saccharomyces cerevisiae

Spleißen ist neben 5‘-Capping und 3‘-Polyadenylierung ein wichtiger Punkt im Lauf der Prozessierung eukaryotischer prä-mRNA. Nichtkodierende Introns werden von der prä-mRNA entfernt und kodierende Exons fusioniert. Doch eine prä-mRNA kann in weitaus mehr als einem Protein resultieren, was durch alternatives Spleißen ermöglicht wird. Über 90 % aller humanen Proteinkodierenden Gene unterstehen diesem Prozess, der zelltyp- bzw. gewebs- und differenzierungsspezifisch ist. Signal-induziertes alternatives Spleißen ist eine Möglichkeit, die Genexpression in Anpassung an Änderungen im physiologischen Umfeld zu regulieren. Der Prozess der T-Zell Aktivierung ist diesbezüglich ein Modelsystem, in dem weit über 100 Exons als alternativ gespleißt identifiziert werden konnten. Wie diese Änderungen im Spleiß- Verhalten zur Funktionalität aktivierter T-Zellen beitragen, konnte jedoch noch nicht hinreichend gezeigt werden. Die T-Zell Aktivierung ist ein komplexer und dynamischer Prozess, der in der aktivierten Zelle u.a. zu einer stark erhöhten Produktion und Sekretion von Effektormolekülen wie Zytokinen und Zytotoxinen führt. Hierfür wird angenommen, dass sich der Sekretionsapparat an die höhere Transportlast anpasst. Wie dies geschieht, ist indes nicht bekannt. Proteinsekretion beginnt an ribosomfreien Stellen des ER, den ER exit sites (ERES), wo neu synthetisierte Proteine in COPII-Vesikel für ihren Transport verpackt werden. Sec16 ist ein peripheres Membranprotein an den ERES und maßgeblich in den Prozess der Vesikelbildung und –abschnürung involviert. Ein in Metazoen konservierter Bereich von ca. 220 Aminosäuren im C-Terminus des Proteins, die C-Terminale konservierte Domäne (CTD) wird u.a. von zwei Exons kodiert (E29 und E30), die nach T-Zell-Aktivierung alternativ gespleißt werden. Diese Arbeit kombiniert biochemische und zellbiologische Methoden, um die funktionelle Relevanz Sec16 Spleißens in der T-Zell Aktivierung zu untersuchen. Initiale Experimente zeigen, dass aktivierte T-Zellen eine erhöhte Sekretionseffizienz aufweisen, die möglicherweise durch eine erhöhte Anzahl an COPII-Vesikeln ermöglicht wird. Durch Manipulation des Spleißverhaltens in einer humanen T-Zell Linie und Etablierung Sec16E29 defizienter Hek293 Zellen mittels Crispr/Cas9 wird gezeigt, dass alternatives Sec16 Spleißen für die Zunahme an COPII-Vesikeln und der Transporteffizienz notwendig ist. Dies wird durch Versuche mit primären humanen PBMCs unterstrichen. Coimmunopräzipitationen belegen eine differenzielle Interaktion der alternativen Sec16 CTDs mit anderen COPII-Proteinen. Zusätzlich zeigen FRAP-Experimente, dass sich der COPII Turnover in Abhängigkeit von Sec16 Spleißen verändert. Diese Arbeit beschreibt die Funktionalität eines Signal- induzierten Spleißvorgangs und schlägt eine neue Form der Regulation im COPII- vermittelten sekretorischen Apparat vor., Beside 5‘-capping and 3‘-polyadenylation, splicing is one major step during eukaryotic pre-mRNA processing. Noncoding introns are removed from the pre- mRNA and coding exons are fused. Due to alternative splicing one single pre- mRNA can result in far more than one protein. Over 90 % of the human protein coding genes undergo this process which acts in a cell type, tissue and differentiation dependent manner. Signal-induced alternative splicing is one possible mechanism to control gene expression due to changes in cellular conditions. In response to T-cell activation hundreds of exons were shown to be alternatively spliced, however, how these splicing switches impact on cellular functionality is largely unknown. T-cell activation is a complex and dynamic process leading, amongst others, to dramatic changes in the production and secretion of effector molecules. Therefore, an increase of the protein secretion capacity seems required. However, how this is achieved remains unknown. Protein secretion starts at special sites of the ER, so called ER exit sites (ERES), where newly synthesized proteins are packed in COPII-coated vesicles for further transport. Sec16 is a peripheral membrane protein at these ERES and necessary for vesicle formation and budding. A part of approximately 220 amino acids in the C-terminal part of the protein, the C-terminal conserved domain (CTD), which is conserved in metazoans, is amongst others encoded by two exons (E29 and E30) that are alternatively spliced upon T-cell activation. This work combines biochemical and cell biological methods to analyse the functional relevance of Sec16 alternative splicing upon T-cell activation. Initial experiments show that activated T-cells have a higher secretion efficiency that might be facilitated by a higher number of COPII coated vesicles. Manipulation of the splicing pattern in a human T-cell line and generation of Sec16E29 deficient Hek293 cells using Crispr/Cas9 show that splicing of Sec16 is required for the increase of COPII coated vesicles and the higher export efficiency. This is underlined by experiments using primary human PBMCs. Coimmunoprecipitations prove a differential interaction of the alternative Sec16-CTDs with other COPII proteins. Additionally FRAP experiments show that COPII Turnover is altered due to Sec16 alternative splicing. This work describes the functional impact of a signal induced splicing switch and provides a new regulatory mechanism for COPII mediated protein secretion.

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.

The endoplasmic reticulum (ER) represents the gateway for intracellular trafficking of membrane proteins, soluble cargoes and lipids. In all eukaryotes, the best described mechanism of exiting the ER is via COPII-coated vesicles, which transport both membrane proteins and soluble cargoes to the cis-Golgi. The vacuole, together with the plasma membrane, is the most distal point of the secretory pathway, and many vacuolar proteins are transported from the ER through intermediate compartments. However, past results and recent findings demonstrate the presence of alternative transport routes from the ER towards the tonoplast, which are independent of Golgi- and post-Golgi trafficking. Moreover, the transport mechanism of the vacuolar proton pumps VHA-a3 and AVP1 challenges the current model of vacuole biogenesis, pointing to the endoplasmic reticulum for being the main membrane source for the biogenesis of the plant lytic compartment. This review gives an overview of the current knowledge on the transport routes towards the vacuole and discusses the possible mechanism of vacuole biogenesis in plants.

Les récepteurs couplés aux protéines G (RCPGs) forment la plus grande et la plus diversifiée des familles de protéines localisées à la surface cellulaire et responsables de la transmission de signaux à l’intérieur des cellules. D’intenses recherches effectuées au cours des trente dernières années ont mené à l’identification de dizaines de protéines interagissant avec les RCPGs et contrôlant la signalisation, la désensibilisation, l’internalisation et la dégradation de ces importantes cibles pharmacologiques. Contrairement aux processus régulant l’activité des récepteurs à partir de la membrane plasmique, les mécanismes moléculaires contrôlant la biosynthèse des RCPGs dans le reticulum endoplasmique (RE) et leur transport jusqu’à la surface cellulaire sont très peu caractérisés. Une meilleure compréhension de ces processus nécessite l’identification de la machinerie protéique responsable de la maturation des RCPGs. Un crible protéomique basé sur le transfert d’énergie de résonance de bioluminescence (BRET), qui permet la mesure d’interactions protéiques dans les cellules vivantes, a mené à l’identification de plusieurs nouvelles protéines localisées dans la voie de sécrétion et interagissant potentiellement avec les RCPGs. Ces protéines étant localisées dans les compartiments cellulaires (reticulum endoplasmique et appareil de Golgi) responsables de la synthèse, du repliement adéquat et du transport à la membrane plasmique des récepteurs, il est très probable qu’elles soient impliquées dans le contrôle de l’expression des RCPGs à la surface cellulaire. La caractérisation de l’homologue humain de cornichon 4 (CNIH4), un nouvel intéracteur des RCPGs identifié dans le crible, a démontré que cette protéine localisée dans les compartiments précoces de la voie de sécrétion (RE et ERGIC) interagit de façon sélective avec les RCPGs. De plus, la suppression de l’expression endogène de cette protéine préalablement non-caractérisée, diminue le transport à la membrane plasmique d’un récepteur, indiquant que CNIH4 influence positivement l’export des RCPGs du RE. Ceci est supporté par l’observation que la surexpression de CNIH4 à de faibles niveaux favorise la maturation d’un récepteur mutant normalement retenu dans le RE. Nous avons également pu démontrer que CNIH4 est associée à la protéine Sec23, une des composantes de l’enveloppe des vésicules COPII qui sont responsables du transport des protéines du RE vers le Golgi, suggérant que CNIH4 pourrait favoriser le recrutement des récepteurs dans ces vésicules. La surexpression de CNIH4 à de très hauts niveaux provoque également la rétention intracellulaire des récepteurs. Cet effet dominant négatif pourrait être causé par la titration d’un autre facteur d’export des RCPGs. Une deuxième étude a permis de révéler que la protéine transmembranaire 9 (TMEM9), un nouvel intéracteur des RCPGs également identifié dans le crible, interagit sélectivement avec les récepteurs et avec CNIH4. La surexpression de cette protéine aux fonctions précédemment inconnues, rétablit le transport normal d’un récepteur en présence de CNIH4 surexprimée. De plus, la co-expression de TMEM9 potentialise la capacité de CNIH4 à augmenter la maturation d’un récepteur mutant normalement retenu dans le RE, suggérant que ces deux protéines forment un complexe régulant la maturation des RCPGs. Au cours de cette thèse, de nouvelles protéines interagissant avec les RCPGs et contrôlant leur expression à la membrane plasmique ont donc été identifiées, permettant une meilleure compréhension des mécanismes régulant le transport des récepteurs du RE à la surface cellulaire., G protein coupled receptors (GPCR) form the largest and most diversified family of cell-surface receptors responsible for signal transduction inside the cells. Extensive research over the last thirty years have led to the identification of multiple proteins interacting with GPCRs and controlling the signalisation, desensitization, internalization and degradation of these important pharmaceutical targets. In contrast to the processes regulating GPCR activity at the plasma membrane, the molecular mechanisms controlling GPCR biogenesis in the endoplasmic reticulum (ER) and their transport to the cell-surface are poorly characterized. The identification of the proteins regulating GPCR maturation is essential in order to understand how receptors are expressed at the plasma membrane. A proteomic screen based on bioluminescence resonance energy transfer (BRET), which allows for the detection of protein-protein interaction in living cells, led to the identification of several potential novel GPCR interactors localized in the secretory pathway. Since the cellular compartments where these proteins are localized are responsible for the synthesis, proper folding and transport to the plasma membrane of the receptors, it is highly probable that they are involve in regulating GPCR cell-surface expression. The characterization of the human cornichon homolog 4 (CNIH4), a novel GPCR interactor identified in the screen, showed that this protein localized in the early secretory pathway (ER and ERGIC), selectively interacts with GPCRs. Knockdown of the endogenous expression of this previously uncharacterized protein led to a decrease in the cell-surface expression of a receptor indicating that CNIH4 has a positive function in the ER export of GPCR. Supporting this, over-expression of CNIH4 at low levels increased the maturation of a mutant receptor normally retained in the ER. Moreover, CNIH4 interacts with Sec23, a component of the inner coat of COPII vesicles which transport proteins from the ER to the Golgi apparatus, suggesting that CNIH4 could recruit GPCRs in these vesicles. CNIH4 over-expression at very high levels also resulted in the intracellular trapping of the receptors. This dominant negative effet could be caused by the titration of another component of the GPCR export process. Another study showed that the transmembrane protein 9 (TMEM9), a novel GPCR interactor also identified in the screen, selectively interacts with GPCRs and CNIH4. Over-expression of this protein of previously unknown function restored normal receptor trafficking in presence of over-expressed CNIH4. Morevover, co-expression of TMEM9 potentialized CNIH4 ability to increase the maturation of a mutant receptor normally retained in the ER, suggesting that these proteins form a complex regulating GPCR maturation. During this thesis, novel GPCR interacting proteins controlling receptor expression at the plasma membrane were identified, allowing for a better understanding of the mechanisms controlling receptor trafficking from the ER to the cell-surface.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.