ABSTRACT
ß-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of ß-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of ß-1,6-glucan by ß-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel ß-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50â and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-ß-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of ß-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: ⢠ß-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. ⢠ß-1,6-Glucanase is an antifungal protein with significant potential applications. ⢠FlGlu30 is the first reported ß-1, 6-glucanase derived from Flavobacterium.
Subject(s)
Antifungal Agents , Cell Wall , Escherichia coli , Flavobacterium , Glycoside Hydrolases , Flavobacterium/enzymology , Flavobacterium/genetics , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hydrolysis , Antifungal Agents/pharmacology , Antifungal Agents/metabolism , Cell Wall/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Glucans/metabolism , Hydrogen-Ion Concentration , beta-Glucans/metabolism , Cloning, Molecular , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature , Substrate Specificity , PolysaccharidesABSTRACT
Ulcerative colitis (UC) is characterized by chronic inflammation and ulceration of the intestinal inner lining, resulting in various symptoms. Sea buckthorn berries contain a bioactive compound known as sea buckthorn polysaccharide (SBP). However, the precise mechanisms underlying the impact of SBP on UC remain unclear. In this study, we investigated the effects of pretreatment with SBP on colitis induced by DSS. Our findings demonstrate that SBP pretreatment effectively reduces inflammation, oxidative stress, and intestinal barrier damage associated with colitis. To further elucidate the role of SBP-modulated gut microbiota in UC, we performed fecal microbiota transplantation (FMT) on DSS-treated mice. The microbiota from SBP-treated mice exhibits notable anti-inflammatory and antioxidant effects, improves colonic barrier integrity, and increases the abundance of beneficial bacteria, as well as enhancing SCFA production. Collectively, these results strongly indicate that SBP-mediated amelioration of colitis is attributed to its impact on the gut microbiota, particularly through the promotion of SCFA-producing bacteria and subsequent elevation of SCFA levels. This study provides compelling evidence supporting the efficacy of pre-emptive SBP supplementation in alleviating colitis symptoms by modulating the gut microbiota, thereby offering novel insights into the potential of SBP as a regulator of the gut microbiota for colitis relief.
Subject(s)
Gastrointestinal Microbiome , Hippophae , Polysaccharides , Animals , Hippophae/chemistry , Polysaccharides/pharmacology , Gastrointestinal Microbiome/drug effects , Mice , Colitis/drug therapy , Colitis/chemically induced , Colitis/microbiology , Colitis, Ulcerative/microbiology , Colitis, Ulcerative/drug therapy , Disease Models, Animal , Male , Mice, Inbred C57BL , Oxidative Stress/drug effects , Fecal Microbiota Transplantation , Colon/drug effects , Colon/microbiology , Colon/metabolism , Dextran Sulfate , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Fruit/chemistry , Fatty Acids, Volatile/metabolismABSTRACT
Endocytosis and autophagy are the main pathways to deliver cargoes in vesicles and autophagosomes, respectively, to vacuoles/lysosomes in eukaryotes. Multiple positive regulators but few negative ones are reported to regulate the entry of vesicles and autophagosomes into vacuoles/lysosomes. In yeast, the Rab5 GTPase Vps21 and the ESCRT (endosomal sorting complex required for transport) are positive regulators in endocytosis and autophagy. During autophagy, Vps21 regulates the ESCRT to phagophores (unclosed autophagosomes) to close them. Phagophores accumulate on vacuolar membranes in both vps21∆ and ESCRT mutant cells under a short duration of nitrogen starvation. The vacuolar transport chaperon (VTC) complex proteins are recently found to be negative regulators in endocytosis and autophagy. Phagophores in vps21∆ cells are promoted to enter vacuoles when the VTC complex proteins are absent. Phagophores are easily observed inside vacuoles when any of these VTC complex proteins (Vtc1, 2, 4, 5) are removed. However, it is unknown whether the removal of VTC complex proteins will also promote the entry of phagophores into vacuoles in ESCRT mutant cells under the same conditions. Snf7 is a core subunit of ESCRT subcomplex III (ESCRT-III), and phagophores accumulate in snf7∆ cells under a short duration of nitrogen starvation. We used green fluorescence protein (GFP) labeled Atg8 to display phagophores and FM4-64-stained or Vph1-GFP-labeled membrane structures to show vacuoles, then examined fluorescence localization and GFP-Atg8 degradation in snf7∆ and snf7∆vtc4∆ cells. Results showed that Vtc4 depletion promoted the entry of phagophores in snf7∆ cells into vacuoles as it did for vps21∆ cells, although the promotion level was more obvious in vps21∆ cells. This observation indicates that the VTC complex proteins may have a widespread role in negatively regulating cargos to enter vacuoles in yeast.
ABSTRACT
Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and fusing with lysosomes/vacuoles, after which the materials are degraded for recycling purposes. Autophagy is associated with increased cell survival under different stress conditions. AP-lysosome/vacuole fusion is a critical step in autophagy. Some mutant cells can accumulate phagophores under autophagy-induction conditions. Autophagy is interrupted when accumulated phagophores cannot fuse with lysosomes/vacuoles, resulting in a significant decrease in cell survivability. However, phagophore-lysosome/vacuole fusion has been reported in related mammalian cells and yeast mutant cells. This observation indicates that it is possible to restore a partial autophagy process after interruption. Furthermore, these findings indicate that phagophore closure is not a prerequisite for its fusion with the lysosome/vacuole in the mutant cells. The phagophore-lysosome/vacuole fusion strategy can significantly rescue defective autophagy due to failed phagophore closure. This commentary discusses the fusion of phagophores and lysosomes/vacuoles and implications of such fusion events.Abbreviations: AB: autophagic body; AL: autolysosome; AP: autophagosome; ATG: autophagy related; EM: electron microscopy; ESCRT: endosomal sorting complex required for transport; ET: electron tomography; FIB: focus ion beam; IM: inner membrane; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; OM; outer membrane; STX17: syntaxin 17; TEM: transmission electron microscopy; TM: transmembrane domain; Vps: vacuolar protein sorting; WT: wild-type.
Subject(s)
Autophagosomes , Vacuoles , Animals , Autophagosomes/metabolism , Vacuoles/metabolism , Saccharomyces cerevisiae/genetics , Autophagy/genetics , Lysosomes/metabolism , Membrane Fusion , MammalsABSTRACT
It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.
Subject(s)
Saccharomyces cerevisiae Proteins , Vacuoles , Autophagosomes/metabolism , Autophagy/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Hydrolases/metabolism , Molecular Chaperones/metabolism , Nitrogen/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sirolimus/metabolism , Sirolimus/pharmacology , Vacuoles/genetics , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.
Subject(s)
Autophagosomes , Saccharomyces cerevisiae Proteins , Animals , Autophagosomes/metabolism , Autophagy , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Endopeptidases/metabolism , Mammals/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
Autophagy-related gene (Atg) proteins are key players in autophagy. Some proteins that function in vesicle trafficking and lipid metabolism are also involved in autophagy. The SPO14 in yeast, which encodes phospholipase D (PLD), is involved in membrane trafficking and plays a vital role in sporulation during meiosis. Crosstalk has been identified between autophagy and sporulation. Although the PLD is required for macroautophagy in mammals, its role in yeast macroautophagy remains unclear. We observed that Spo14 is not required for macroautophagy in yeast and that it is dispensable for Atg8 lipidation, which plays an important role in phagophore extension. Our results also revealed that green fluorescent protein (GFP)-Atg8 degradation is not completely blocked in atg1Δ/atg1Δ cells under sporulation condition. Therefore, Spo14 is not required for macroautophagy in yeast.
Subject(s)
Autophagy , Phospholipase D , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Animals , Autophagy/genetics , Autophagy/physiology , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Macroautophagy , Mammals , Meiosis , Phospholipase D/genetics , Phospholipase D/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Protein-protein interactions are important for physiology performance. Green fluorescent protein (GFP) is a widely used protein tag to show protein localization in vivo. GFP binding protein (GBP) is a specific domain with high affinity to GFP. A novel technique with GBP fused protein X tagged with red fluorescence protein binding to GFP of GFP fused protein Y to establish a close association for proteins X and Y independently from other proteins has recently been developed. It is found that the interaction and colocalization between Snf7 and Atg17 is impaired in Saccharomyces cerevisiae vps21Δ cells, which are defective in autophagy. In order to determine whether the interaction between Snf7 and Atg17 is important for autophagy, we forced the interaction between Snf7 and Atg17 through GBP-GFP binding. Snf7-GBP-mCherry and/or GFP-Atg17 tagged wild-type and vps21Δ cells were compared for autophagy process under starvation by determining the maturation of proprotein of Ape1 (prApe1). Our results showed that the defective autophagy in vps21Δ cells was significantly suppressed when both Snf7-GBP-mCherry and GFP-Atg17 were installed. Our results indicate that the GBP-GFP nanotrap technique is a powerful tool to restore colocalization/interaction in vivo and the Snf7-Atg17 interaction is important for yeast autophagy.
Subject(s)
Autophagy , Autophagy/genetics , Autophagy-Related Proteins/metabolism , Endosomal Sorting Complexes Required for Transport , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding ProteinsABSTRACT
Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.
Subject(s)
Autophagosomes , Saccharomyces cerevisiae Proteins , Animals , Autophagy , Endosomal Sorting Complexes Required for Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Adenosine Triphosphatases , Autophagosomes , Autophagy-Related Proteins/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Mitophagy , Receptors, Cytoplasmic and Nuclear , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive. RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response. CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.
Subject(s)
Autophagy , Cell Membrane/physiology , Diglycerides/metabolism , Homeostasis , Saccharomyces cerevisiae/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Protein TransportABSTRACT
Lipid droplets (LDs) are cytosolic fat storage organelles that play roles in lipid metabolism, trafficking and signaling. Breakdown of LDs in Saccharomyces cerevisiae is mainly achieved by lipolysis and lipophagy. In this study, we found that the endosomal sorting complex required for transport (ESCRT) in S. cerevisiae negatively regulated the turnover of a LD marker, Erg6, under both simplified glucose restriction (GR) and acute glucose restriction (AGR) conditions by monitoring the localization and degradation of Erg6. Loss of Vps27, Snf7 or Vps4, representative subunits of the ESCRT machinery, facilitated the delivery of Erg6-GFP to vacuoles and its degradation depending on the lipophagy protein Atg15 under simplified GR. Additionally, the lipolysis proteins Tgl3 and Tgl4 were also involved in the enhanced vacuolar localization and degradation of Erg6-GFP in vps4Δ cells. Furthermore, we found that Atg14, which is required for the formation of putatively liquid-ordered (Lo) membrane domains on the vacuole that act as preferential internalization sites for LDs, abundantly localized to vacuolar membranes in ESCRT mutants. Most importantly, the depletion or overexpression of Atg14 correspondingly abolished or promoted the observed Erg6 degradation in ESCRT mutant cells. We propose that Atg14 together with other proteins promotes Erg6 degradation in ESCRT mutant cells under specific glucose restriction conditions. These results shed new light on the regulation of ESCRT on LD turnover.
Subject(s)
Endosomal Sorting Complexes Required for Transport , Glucose , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Adenosine Triphosphatases , Endosomal Sorting Complexes Required for Transport/metabolism , Glucose/metabolism , Methyltransferases , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The macroautophagy/autophagy pathway includes successive steps of phagophore assembly structure formation, phagophore expansion, autophagosome (AP) closure and AP fusion with the lysosome/vacuole. Although information about regulators, factors and molecular mechanisms important for early and late steps of autophagy is abundant, information about AP closure is scarce. In 2017, we reported that the Vps21/RAB5 GTPase module regulates AP closure in yeast. In a recent paper, we show that Vps21 regulates the recruitment of ESCRT to APs to catalyze their closure by controlling an Atg17-Snf7 interaction. Thus, we identify a regulator, a factor, and a molecular mechanism important for AP closure. Abbreviations: AP: Autohagosome; Atg: autophagy-related gene; ESCRT: the endosomal sorting complex required for transport; ILVs: intralumenal vescicles.
Subject(s)
Autophagy , Saccharomyces cerevisiae Proteins , Autophagosomes , Endosomal Sorting Complexes Required for Transport , Endosomes , VacuolesABSTRACT
Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9-domain-containing protein, VARP-like 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9-domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9-domain-containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9-domain-containing proteins, Vps9, Muk1, and Vrl1, all co-localized with Atg8 on autophagosomes in cells blocked in any late step of starvation-induced autophagy, with Vrl1 most often co-localizing with Atg8. A small portion (<25%) of these VPS9-domain-containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy-dependent and -independent routes.
Subject(s)
Autophagy-Related Proteins/metabolism , Autophagy , Guanine Nucleotide Exchange Factors/metabolism , Protein Domains , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolism , Autophagy-Related Protein 8 Family/metabolism , Protein Transport , Vacuoles/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.
Subject(s)
Autophagosomes/physiology , Autophagy , Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , rab5 GTP-Binding Proteins/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Intracellular Membranes , Lysosomes/metabolism , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , rab5 GTP-Binding Proteins/geneticsABSTRACT
Snf7 is the core subunit protein of the yeast endosomal sorting complex required for transport (ESCRT) complex, which plays important roles in endocytosis and autophagy. In this study, we characterized MoSnf7 in Magnaporthe oryzae, a homolog of yeast Snf7, the core protein of ESCRT-III subcomplex. Like Snf7, MoSnf7 also localizes next to the vacuoles. Deletion of MoSNF7 resulted in significant decrease in vegetative growth and pathogenicity. Further analyses of ΔMosnf7 mutants showed that they were defective in endocytosis, sexual and asexual development, turgor pressure maintenance of appressorium at hyphal tips, and cell wall integrity. Additional assays for the localization and degradation of GFP-MoAtg8 in ΔMosnf7 mutants showed that they were defective in autophagy pathway. Based on the roles of yeast Snf7 in endocytosis and autophagy, we propose that the decreased vegetative growth and pathogenicity of ΔMosnf7 rice blast fungus M. oryzae, was partly due to the conservative roles of MoSnf7 in vesicle trafficking and autophagy pathway.
Subject(s)
Magnaporthe/genetics , Oryza/microbiology , Plant Diseases/genetics , Spores, Fungal/genetics , Autophagy/genetics , Cell Wall/metabolism , Endocytosis/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Fungal Proteins/genetics , Magnaporthe/pathogenicity , Oryza/genetics , Plant Diseases/microbiology , Saccharomyces cerevisiae Proteins/genetics , Spores, Fungal/growth & developmentABSTRACT
Honokiol (HNK), an important medicinal component of Magnolia officinalis, is reported to possess pharmacological activities against a variety of diseases. However, the molecular mechanisms of HNK medicinal functions are not fully clear. To systematically study the mechanisms of HNK action, we screened a yeast mutant library based on the conserved nature of its genes among eukaryotes. We identified genes associated with increased resistance or sensitivity to HNK after mutation. After functional classification of these genes, we found that most HNK-resistant strains in the largest functional category were petites with mutations in mitochondrial genes, indicating that mitochondria were related to HNK resistance. Additional analysis showed that resistance of petite mutants to HNK was associated with upregulation of the ATP-binding cassette transporter Pdr5, which pumps out HNK. We also found that several HNK-sensitive mitochondria mutants were not petites, and had larger lipid droplets (LDs). Furthermore, HNK treatment on wild-type yeast cells seemed to disrupt mitochondrial morphology, induced triacylglycerol synthesis, and generated supersized LDs surrounded by mitochondria and endoplasmic reticulum (ER). These changes are also applied to atp7Δ mutant if no carbon resource was available. These results suggested that HNK treatment partly impaired normal mitochondrial function to form larger LDs by altering lipid metabolism.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Biphenyl Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Genes, Fungal , Lignans/pharmacology , Lipid Metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae Proteins/genetics , Drug Resistance/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/geneticsABSTRACT
In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.
Subject(s)
Autophagosomes/metabolism , Endocytosis/genetics , Protein Transport/genetics , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/genetics , Autophagy/genetics , Autophagy-Related Proteins/genetics , Endosomes/genetics , Lysosomes/genetics , Phosphatidylinositol 3-Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/geneticsABSTRACT
Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the cis-Golgi localized t-SNARE protein Sed5 plays a role in endoplasmic reticulum (ER)-Golgi and intra-Golgi vesicular transport. We report that during autophagy, sed5-1 mutant cells could not properly transport Atg8 to the PAS, resulting in multiple Atg8 dots being dispersed into the cytoplasm. Some dots were trapped in the Golgi apparatus. Sed5 regulates the antero-grade trafficking of Atg9-containing vesicles to the PAS by participating in the localization of Atg23 and Atg27 to the Golgi apparatus. Furthermore, we found that overexpression of SFT1 or SFT2 (suppressor of sed5 ts) rescued the autophagy defects in sed5-1 mutant cells. Our data suggest that Sed5 plays a novel role in autophagy, by regulating the formation of Atg9-containing vesicles in the Golgi apparatus, and the genetic interaction between Sft1/2 and Sed5 is essential for autophagy.
Subject(s)
Autophagy-Related Proteins/genetics , Autophagy/genetics , Membrane Proteins/genetics , Protein Transport/genetics , Qa-SNARE Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Autophagosomes/metabolism , Autophagy-Related Proteins/metabolism , COP-Coated Vesicles/genetics , COP-Coated Vesicles/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Gene Expression Regulation, Fungal/genetics , Protein Binding , Qc-SNARE Proteins/genetics , Saccharomyces cerevisiae/genetics , Vesicular Transport Proteins/geneticsABSTRACT
Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth.