RESUMO
Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.
Assuntos
Colite Ulcerativa , Fucosiltransferases , Animais , Camundongos , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Fucosiltransferases/genética , Inflamação , Mucina-2/genética , Mucina-2/metabolismo , Células HT29RESUMO
Nutrient deprivation triggers the release of signal-sequence-lacking Acb1 and the antioxidant superoxide dismutase 1 (SOD1). We now report that secreted SOD1 is functionally active and accompanied by export of other antioxidant enzymes such as thioredoxins (Trx1 and Trx2) and peroxiredoxin Ahp1 in a Grh1-dependent manner. Our data reveal that starvation leads to production of nontoxic levels of reactive oxygen species (ROS). Treatment of cells with N-acetylcysteine (NAC), which sequesters ROS, prevents antioxidants and Acb1 secretion. Starved cells lacking Grh1 are metabolically active, but defective in their ability to regrow upon return to growth conditions. Treatment with NAC restored the Grh1-dependent effect of starvation on cell growth. In sum, starvation triggers ROS production and cells respond by secreting antioxidants and the lipogenic signaling protein Acb1. We suggest that starvation-specific unconventional secretion of antioxidants and Acb1-like activities maintain cells in a form necessary for growth upon their eventual return to normal conditions.
Assuntos
Antioxidantes/metabolismo , Proteínas de Transporte/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
It is usually assumed that eukaryotic cells secrete only proteins that contain a signal sequence for Sec61 mediated translocation into the lumen of endoplasmic reticulum (ER). Surprisingly however, many proteins, such as superoxide dismutase (SOD)1, acyl-CoA binding protein (Acb1), interleukin 1ß, fibroblast growth factor 2 and the adipokine Unpaired2, to name a few, are secreted even though they lack a signal sequence. The discovery that these proteins are secreted has presented a new challenge and we describe here a common pathway by which SOD1 and Acb1 are specifically secreted upon nutrient starvation. Their secretion follows a type III unconventional pathway, requiring the exposure of a di-acidic motif, which we propose promotes their capture into a membrane compartment called CUPS (compartment for unconventional protein secretion). We suggest that CUPS, composed of membranes derived from the Golgi apparatus and endosomes, serves as a major sorting station prior to release of SOD1 and Acb1 into the extracellular space. The trafficking of these signal sequence lacking proteins therefore has functional similarities to conventional protein secretion in that they rely on membrane bounded compartments for their sorting and transport, but bypass the need of Sec61 for translocating into the ER and COPII and COPI for their intracellular transfers. This review is part of a Special Issue of SCDB on "unconventional protein secretion" edited by Walter Nickel and Catherine Rabouille.
Assuntos
Proteínas de Transporte/metabolismo , Nutrientes/metabolismo , Transporte Proteico/fisiologia , HumanosRESUMO
The nutrient starvation-specific unconventional secretion of Acb1 in Saccharomyces cerevisiae requires ESCRT-I, -II, and -III and Grh1. In this study, we report that another signal sequence lacking cytoplasmic protein, superoxide dismutase 1 (SOD1), and its mutant form linked to amyotrophic lateral sclerosis (ALS), is also secreted by yeast upon nutrient starvation in a Grh1- and ESCRT-I-, -II-, and -III-dependent process. Our analyses reveal that a conserved diacidic motif (Asp-Glu) in these proteins is necessary for their export. Importantly, secretion of wild-type human SOD1 and the ALS-linked mutant in human cells also require the diacidic residues. Altogether, these findings reveal information encoded within the cytoplasmic proteins required for their unconventional secretion and provide a means to unravel the significance of the cytoplasmic versus the secreted form of mutant SOD1 in the pathology of ALS. We also propose how cells, based on a signal-induced change in cytoplasmic physiology, select a small pool of a subset of cytoplasmic proteins for unconventional secretion.
RESUMO
The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Deleção de Genes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Upon starvation, Grh1, a peripheral membrane protein located at endoplasmic reticulum (ER) exit sites and early Golgi in Saccharomyces cerevisiae under growth conditions, relocates to a compartment called compartment for unconventional protein secretion (CUPS). Here we report that CUPS lack Golgi enzymes, but contain the coat protein complex II (COPII) vesicle tethering protein Uso1 and the Golgi t-SNARE Sed5. Interestingly, CUPS biogenesis is independent of COPII- and COPI-mediated membrane transport. Pik1- and Sec7-mediated membrane export from the late Golgi is required for complete assembly of CUPS, and Vps34 is needed for their maintenance. CUPS formation is triggered by glucose, but not nitrogen starvation. Moreover, upon return to growth conditions, CUPS are absorbed into the ER, and not the vacuole. Altogether our findings indicate that CUPS are not specialized autophagosomes as suggested previously. We suggest that starvation triggers relocation of secretory and endosomal membranes, but not their enzymes, to generate CUPS to sort and secrete proteins that do not enter, or are not processed by enzymes of the ER-Golgi pathway of secretion.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Secretórias/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Meios de Cultura , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
The Sec14 protein domain is a conserved tertiary structure that binds hydrophobic ligands. The Sec14 protein from Saccharomyces cerevisiae is essential with studies of S. cerevisiae Sec14 cellular function facilitated by a sole temperature sensitive allele, sec14(ts). The sec14(ts) allele encodes a protein with a point mutation resulting in a single amino acid change, Sec14(G266D). In this study results from a genome-wide genetic screen, and pharmacological data, provide evidence that the Sec14(G266D) protein is present at a reduced level compared to wild type Sec14 due to its being targeted to the proteosome. Increased expression of the sec14(ts) allele ameliorated growth arrest, but did not restore the defects in membrane accumulation or vesicular transport known to be defective in sec14(ts) cells. We determined that trafficking and localization of two well characterized lipid raft resident proteins, Pma1 and Fus-Mid-GFP, were aberrant in sec14(ts) cells. Localization of both lipid raft proteins was restored upon increased expression of the sec14(ts) allele. We suggest that a major function provided by Sec14 is trafficking and localization of lipid raft proteins.
Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Primers do DNA/genética , Deleção de Genes , Microscopia Eletrônica , Mutação de Sentido Incorreto/genética , Proteínas de Transferência de Fosfolipídeos/genética , Plasmídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMO
The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca(2+) ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H(+) ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Rede trans-Golgi/metabolismo , Fatores de Despolimerização de Actina/genética , Western Blotting , Catepsina A/genética , Catepsina A/metabolismo , Membrana Celular/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Endocitose , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Microscopia de Fluorescência , Mutação , Transporte Proteico , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismoRESUMO
The endoplasmic reticulum (ER)-Golgi-independent, unconventional secretion of Acb1 requires many different proteins. They include proteins necessary for the formation of autophagosomes, proteins necessary for the fusion of membranes with the endosomes, proteins of the multivesicular body pathway, and the cell surface target membrane SNARE Sso1, thereby raising the question of what achieves the connection between these diverse proteins and Acb1 secretion. In the present study, we now report that, upon starvation in Saccharomyces cerevisiae, Grh1 is collected into unique membrane structures near Sec13-containing ER exit sites. Phosphatidylinositol 3 phosphate, the ESCRT (endosomal sorting complex required for transport) protein Vps23, and the autophagy-related proteins Atg8 and Atg9 are recruited to these Grh1-containing membranes, which lack components of the Golgi apparatus and the endosomes, and which we call a novel compartment for unconventional protein secretion (CUPS). We describe the cellular proteins required for the biogenesis of CUPS, which we believe is the sorting station for Acb1's release from the cells.
Assuntos
Autofagia/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fagossomos , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
A protein known to regulate both lipid metabolism and vesicular transport is the phosphatidylcholine/phosphatidylinositol transfer protein Sec14 of Saccharomyces cerevisiae. Sec14 is thought to globally affect secretion from the trans-Golgi. The results from a synthetic genetic array screen for genes whose inactivation impaired growth of cells with a temperature-sensitive SEC14 allele implied Sec14 regulates transport into and out of the Golgi. This prompted us to examine the role of Sec14 in various vesicular transport pathways. We determined that Sec14 function was required for the route followed by Bgl2, whereas trafficking of other secreted proteins, including Hsp150, Cts1, Scw4, Scw10, Exg1, Cis3, and Ygp1, still occurred, indicating Sec14 regulates specific trans-Golgi export pathways. Upon diminution of Sec14 function, the v-SNARE Snc1 accumulated in endosomes and the trans-Golgi. Its accumulation in endosomes is consistent with Sec14 being required for transport from endosomes to the trans-Golgi. Sec14 was also required for trafficking of Ste3 and the lipophilic dye FM4-64 from the plasma membrane to the vacuole at the level of the endosome. The combined genetic and cell biology data are consistent with regulation of endosome trafficking being a major role for Sec14. We further determined that lipid ligand occupancy differentially regulates Sec14 functions.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endossomos/genética , Complexo de Golgi/genética , Metabolismo dos Lipídeos/fisiologia , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the alpha-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the beta-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The beta-importin Kap95 is a direct regulator membrane synthesis.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Retículo Endoplasmático/metabolismo , Poro Nuclear/metabolismo , Fosfatidilcolinas/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sistema Livre de Células/metabolismo , Colina-Fosfato Citidililtransferase/genética , Retículo Endoplasmático/genética , Modelos Biológicos , Poro Nuclear/genética , Fosfatidilcolinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/genéticaRESUMO
The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer protein Sec14p is required for Golgi apparatus-derived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP-choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of "sec14 bypass" were similar or distinct. The results indicate that inactivation of the CDP-choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described.