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1.
The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens.  相似文献   

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Starving Dictyostelium discoideum cells secrete AcbA, an acyl coenzyme A–binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo.  相似文献   

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In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.  相似文献   

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In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)–binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2–like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.  相似文献   

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Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the Vo domain (Voa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V1Vo assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pHvph1Δ/Δ = 6.8 versus pHvph1Δ/ΔR = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.  相似文献   

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Trichoderma species are opportunistic fungi residing primarily in soil, tree bark and on wild mushrooms. Trichoderma is capable of killing other fungi and penetrating plant roots, and is commonly used as both a biofungicide and inducer of plant defence against pathogens. These fungi also exert other beneficial effects on plants including growth promotion and tolerance to abiotic stresses, primarily mediated by their intimate interactions with roots. In root–microbe interactions (both beneficial and harmful), fungal secreted proteins play a crucial role in establishing contact with the roots, fungal attachment, root penetration and triggering of plant responses. In Trichoderma–root interactions, the sucrose present in root exudates has been demonstrated to be important in fungal attraction. Attachment to roots is mediated by hydrophobin-like proteins, and secreted swollenins and plant cell wall degrading enzymes facilitate internalization of the fungal hyphae. During the early stage of penetration, suppression of plant defence is vital to successful initial root colonisation; this is mediated by small soluble cysteine-rich secreted proteins (effector-like proteins). Up to this stage, Trichoderma's behaviour is similar to that of a plant pathogen invading root structures. However, subsequent events like oxidative bursts, the synthesis of salicylic acid by the plants, and secretion of elicitor-like proteins by Trichoderma spp. differentiate this fungus from pathogens. These processes induce immunity in plants that help counter subsequent invasion by plant pathogens and insects. In this review, we present an inventory of soluble secreted proteins from Trichoderma that might play an active role in beneficial Trichoderma–plant interactions, and review the function of such proteins where known.  相似文献   

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《Autophagy》2013,9(5):650-651
In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.  相似文献   

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The ability to form hyphae in the human pathogenic fungus Candida albicans is a prerequisite for virulence. It contributes to tissue infection, biofilm formation, as well as escape from phagocytes. Cell elongation triggered by human body temperature involves the essential heat shock protein Hsp90, which negatively governs a filamentation program dependent upon the Ras-protein kinase A (PKA) pathway. Tight regulation of Hsp90 function is required to ensure fast appropriate response and maintenance of a wide range of regulatory and signaling proteins. Client protein activation by Hsp90 relies on a conformational change of the chaperone, whose ATPase activity is competitively inhibited by geldanamycin. We demonstrate a novel regulatory mechanism of heat- and Hsp90-dependent induced morphogenesis, whereby the nonreducing disaccharide trehalose acts as a negative regulator of Hsp90 release. By means of a mutant strain deleted for Gpr1, the G protein-coupled receptor upstream of PKA, we demonstrate that elevated trehalose content in that strain, resulting from misregulation of enzymatic activities involved in trehalose metabolism, disrupts the filamentation program in response to heat. Addition of geldanamycin does not result in hyphal extensions at 30 °C in the gpr1Δ/gpr1Δ mutant as it does in wild type cells. In addition, validamycin, a specific inhibitor of trehalase, the trehalose-degrading enzyme, inhibits cell elongation in response to heat and geldanamycin. These results place Gpr1 as a regulator of trehalose metabolism in C. albicans and illustrate that trehalose modulates Hsp90-dependent activation of client proteins and signaling pathways leading to filamentation in the human fungal pathogen.  相似文献   

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In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)–binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.In eukaryotic cells, most secreted proteins rely on the highly conserved secretory pathway for their release into the extracellular space. Signal sequences target them for cotranslational translocation across the ER membrane, and the proteins fold within the ER lumen. The proteins are then transported to and through the Golgi apparatus, sorted, and delivered to the cell surface. The machinery responsible for the secretory pathway is comprised of proteins that collect cargo, form transport vesicles, and help vesicles recognize and fuse at the correct target membranes. A small number of secreted proteins use secretory pathway-independent routes by a process called unconventional secretion (Nickel and Rabouille, 2009). In this issue, Duran et al. and Manjithaya et al. make powerful use of yeast genetics to provide new mechanistic insight into the previously unknown, unconventional route taken by an acyl CoA–binding protein (ACBP) to reach the extracellular space.The simplest pathway for unconventional secretion is that taken by the yeast a-factor mating pheromone. This farnesylated and methylated dodecapeptide is exported by the STE6 gene product that encodes an ATP-binding cassette (ABC) family transporter (Kuchler et al., 1989; McGrath and Varshavsky, 1989). Larger proteins, including FGF2, galectins 1 and 3, a subset of interleukins, and the engrailed homeodomain protein are also unconventional secretory cargoes, but their precise routes of export are unknown (Nickel and Rabouille, 2009). During an inflammatory response, interleukin-1β is somehow translocated from the cytosol into secretory lysosomes for release from cells by a still poorly defined mechanism. Caspase-1 may be required for the unconventional secretion of all of these proteins, suggesting that they may use a common route (Keller et al., 2008).Unconventional secretion of an ACBP was first reported in Dictyostelium discoideum. In this organism, spore formation is activated by release of the 10-kD, ER signal sequence lacking, AcbA protein from prespore cells. Secreted AcbA is proteolyzed extracellularly to produce SDF-2 (spore differentiation factor–2; Anjard et al., 1998). Kinseth et al. (2007) showed that a Golgi-associated protein, GRASP, was required for AcbA release and subsequent SDF-2 production but not for cell growth. Inhibitors of ABC family transporters had no influence on AcbA release. Kinseth et al. (2007) revealed an entirely unexpected role for a Golgi protein in unconventional secretion and a route for AcbA that was distinct from a-factor. A conserved role for GRASP was also later reported for the unconventional secretion of α-integrin at a specific stage of Drosophila melanogaster development (Schotman et al., 2008).Duran et al. (2010) now show that secretion of the Saccharomyces cerevisiae AcbA orthologue, Acb1, also requires the corresponding yeast GRASP orthologue, Grh1. As in D. discoideum, nitrogen starvation triggered Acb1 secretion in a concerted pulse. Genes known to be essential for conventional secretion (SEC23, SEC7, or SEC1) or a-factor release (STE6) were not needed for Acb1 release, confirming that the protein uses an unconventional pathway. However, the SEC18 gene that encodes the NSF ATPase was needed. This ATPase disassembles SNARE proteins and is required for all cellular membrane fusion events.The requirement for starvation suggested that an autophagosome intermediate might be involved. Indeed, mutants impaired in various stages of autophagy were all deficient in Acb1 secretion. Fusion of autophagosomes with the vacuole was not required, but the endosomal t-SNARE, Tlg2, and the plasma membrane t-SNARE, Sso1, were required. Finally, convergence of the autophagosomal and the multivesicular endosome pathways was required.Manjithaya et al. (2010) monitored release of Acb1 from the yeast Pichia pastoris by assaying the generation of an SDF-2–like activity that would trigger sporulation in D. discoideum. As in S. cerevisiae, release from P. pastoris required the GRASP homologue Grh1 and numerous autophagy gene products, in particular, Atg11, which is required for receptor-dependent autophagy (Xie and Klionsky, 2007). Similar to the baker’s yeast findings, a plasma membrane t-SNARE was also implicated.Production of medium chain fatty acyl CoAs was needed for Acb1 secretion from P. pastoris. Manjithaya et al. (2010) propose that Acb1 secretion may require that Acb1 bind its medium-chain acyl CoA substrate. Alternatively, the acyl CoA could be needed to acylate a protein (or proteins) that participates in autophagosomal incorporation of Acb1 protein. Lipid modification and/or binding seem to be a recurring theme for unconventional secretion cargoes (Nickel and Rabouille, 2009) and may contribute to incorporation into nascent autophagosomal structures.These experiments suggest that Acb1 is targeted for selective autophagy, a process that begins with recruitment to a so-called phagophore assembly site (Fig. 1). Phagophores are engulfed by multivesicular endosomes that normally deliver their contents to the yeast vacuole (or lysosomes). In some cases, a subset of multivesicular endosomes fuses with the plasma membrane and releases their contents (Simons and Raposo, 2009; Théry et al., 2009). In these studies, fusion of phagophores with multivesicular endosomes and subsequent fusion of these compartments with the plasma membrane appear to represent the major route of unconventional secretion of ACBPs. The use of specific mutant yeast strains has provided key insight into the specific pathways taken by unusual secretory cargoes. These studies also implicate specific SNARE proteins in the poorly understood, multivesicular endosome release process.Open in a separate windowFigure 1.A model for unconventional secretion of Acb1. Selective autophagy involves cargo collection on the surface of a phagophore membrane (blue). These are engulfed by a multivesicular endosome that fuses with the plasma membrane to release its content. Whether the phagophore is released from an endosomal, lumenal vesicle by lipase action before exocytosis (?) is not known. Duran et al. (2010) and Manjithaya et al. (2010) show that the t-SNARE Sso1 is needed for exocytosis, and fusion with the vacuole is not required.What conserved role does GRASP play? A connection between autophagy and the Golgi complex was recently reported by Itoh et al. (2008), who showed a direct link between the autophagy protein Atg16L1 and the Golgi Rab GTPase Rab33b. We do not yet know the precise origins of the phagophore membrane that participates in unconventional secretion, but roles for GRASP and Rab33b suggest that the Golgi is clearly important for this process. Does GRASP help segregate membrane components needed to form a nascent phagophore? How do ACBPs and other unconventionally secreted substrates actually engage the autophagy machinery? ACBP release involves nitrogen starvation; therefore, is stress important for unconventional secretion, and do other stress signals trigger an autophagic response? Important areas for future research include the identification of such signals, the elucidation of the mechanisms by which these signals are translated into cargo sequestration, and determination of the breadth and diversity of proteins that make use of this unconventional secretory pathway.  相似文献   

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Under specific environmental conditions, the yeast Saccharomyces cerevisiae can undergo a morphological switch to a pseudohyphal growth pattern. Pseudohyphal differentiation is generally studied upon induction by nitrogen limitation in the presence of glucose. It is known to be controlled by several signaling pathways, including mitogen-activated protein kinase, cyclic AMP-protein kinase A (cAMP-PKA), and Snf1 kinase pathways. We show that the alpha-glucoside sugars maltose and maltotriose, and especially sucrose, are more potent inducers of filamentation than glucose. Sucrose even induces filamentation in nitrogen-rich media and in the mep2Δ/mep2Δ ammonium permease mutant on ammonium-limiting medium. We demonstrate that glucose also inhibits filamentation by means of a pathway parallel to the cAMP-PKA pathway. Deletion of HXK2 shifted the pseudohyphal growth pattern on glucose to that of sucrose, while deletion of SNF4 abrogated filamentation on both sugars, indicating a negative role of glucose repression and a positive role for Snf1 activity in the control of filamentation. In all strains and in all media, sucrose induction of filamentation is greatly diminished by deletion of the sucrose/glucose-sensing G-protein-coupled receptor Gpr1, whereas it has no effect on induction by maltose and maltotriose. The competence of alpha-glucoside sugars to induce filamentation is reflected in the increased expression of the cell surface flocculin gene FLO11. In addition, sucrose is the only alpha-glucoside sugar capable of rapidly inducing FLO11 expression in a Gpr1-dependent manner, reflecting the sensitivity of Gpr1 for this sugar and its involvement in rapid sucrose signaling. Our study identifies sucrose as the most potent nutrient inducer of pseudohyphal growth and shows that glucose inactivation of Snf1 kinase signaling is responsible for the lower potency of glucose.  相似文献   

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Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens ΔtliA ΔprtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.  相似文献   

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Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.  相似文献   

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Vivek Malhotra 《The EMBO journal》2013,32(12):1660-1664
The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.  相似文献   

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