A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles

Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube

Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requiresSNAREproteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, theRAB7GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1-Ccz1. We and others recently identified Ykt6 as the autophagosomalSNAREprotein. However, it has not been resolved when and how lipid-anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)-resident Dsl1 complex andCOPII-coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event

Comparative transcriptome analysis reveals the potential mechanism of GA3-induced dormancy release in Suaeda glauca black seeds

Suaeda glauca Bunge produces dimorphic seeds on the same plant, with brown seeds displaying non-dormant characteristics and black seeds exhibiting intermediate physiological dormancy traits. Previous studies have shown that black seeds have a very low germination rate under natural conditions, but exogenous GA3 effectively enhanced the germination rate of black seeds. However, the physiological and molecular mechanisms underlying the effects of GA3 on S. glauca black seeds are still unclear. In this study, transcriptomic profiles of seeds at different germination stages with and without GA3 treatment were analyzed and compared, and the TTF, H2O2, O2–, starch, and soluble sugar contents of the corresponding seed samples were determined. The results indicated that exogenous GA3 treatment significantly increased seed vigor, H2O2, and O2– contents but decreased starch and soluble sugar contents of S. glauca black seeds during seed dormancy release. RNA-seq results showed that a total of 1136 DEGs were identified in three comparison groups and were involved mainly in plant hormone signal transduction, diterpenoid biosynthesis, flavonoid biosynthesis, phenylpropanoid biosynthesis, and carbohydrate metabolism pathway. Among them, the DEGs related to diterpenoid biosynthesis (SgGA3ox1, SgKAO and SgGA2ox8) and ABA signal transduction (SgPP2Cs) could play important roles during seed dormancy release. Most genes involved in phenylpropanoid biosynthesis were activated under GA3 treatment conditions, especially many SgPER genes encoding peroxidase. In addition, exogenous GA3 treatment also significantly enhanced the expression of genes involved in flavonoid synthesis, which might be beneficial to seed dormancy release. In accordance with the decline in starch and soluble sugar contents, 15 genes involved in carbohydrate metabolism were significantly up-regulated during GA3-induced dormancy release, such as SgBAM, SgHXK2, and SgAGLU, etc. In a word, exogenous GA3 effectively increased the germination rate and seed vigor of S. glauca black seeds by mediating the metabolic process or signal transduction of plant hormones, phenylpropanoid and flavonoid biosynthesis, and carbohydrate metabolism processes. Our results provide novel insights into the transcriptional regulation mechanism of exogenous GA3 on the dormancy release of S. glauca black seeds. The candidate genes identified in this study may be further studied and used to enrich our knowledge of seed dormancy and germination

TORC1 determines Fab1 lipid kinase function at signaling endosomes and vacuoles

Acknowledgments: We thank Lars Langemeyer for feedback, all members from the Ungermann lab for discussions, and Kathrin Auffarth, Angela Perz, and Malika Jaquenoud for expert technical assistance. This work was supported by the DFG (UN111/10-1 to C.U.), the Canton of Fribourg (to J.D. and C.D.V.), and the Swiss National Science Foundation (310030_166474/184671 to C.D.V. and 310030_184781 and 316030_177088 to J.D.). Z.C. received support from a travel stipend of the Boehringer Ingelheim Fonds. P.C.M. received additional support from the graduate program of the Collaborative Research Center 944 (SFB 944) and Department of Biology/Chemistry Osnabrück. E.E. received a fellowship of FWO Vlaanderen, Belgium (SB-FWO 1S06419N). Author Contributions: Z.C. and P.C.M. conducted all experiments on Fab1 localization and function; R.H. conducted experiments on development and analysis of the Sch91–183 probe; R.N., Z.H., M.-P.P.-G., and J.D. did the phosphorylation assays and analyses; and E.E. and J.W. conceived and performed the initial Sch9 mapping. T.N. and C.J.S. did the lipid analysis of the mutant alleles. J.G. analyzed microcopy data with Z.C. C.D.V. and C.U. conceived the study and wrote the manuscript with support of J.W.Peer reviewedPublisher PD

Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing

Suppressive Effects on the Immune Response and Protective Immunity to a JEV DNA Vaccine by Co-administration of a GM-CSF-Expressing Plasmid in Mice

As a potential cytokine adjuvant of DNA vaccines, granulocyte-macrophage colony–stimulating factor (GM-CSF) has received considerable attention due to its essential role in the recruitment of antigen-presenting cells, differentiation and maturation of dendritic cells. However, in our recent study of a Japanese encephalitis virus (JEV) DNA vaccine, co-inoculation of a GM-CSF plasmid dramatically suppressed the specific IgG response and resulted in decreased protection against JEV challenge. It is known that GM-CSF has been used in clinic to treat neutropenia for repopulating myeloid cells, and as an adjuvant in vaccine studies; it has shown various effects on the immune response. Therefore, in this study, we characterized the suppressive effects on the immune response to a JEV DNA vaccine by the co-administration of the GM-CSF-expressing plasmid and clarified the underlying mechanisms of the suppression in mice. Our results demonstrated that co-immunization with GM-CSF caused a substantial dampening of the vaccine-induced antibody responses. The suppressive effect was dose- and timing-dependent and likely related to the immunogenicity of the antigen. The suppression was associated with the induction of immature dendritic cells and the expansion of regulatory T cells but not myeloid-derived suppressor cells. Collectively, our findings not only provide valuable information for the application of GM-CSF in clinic and using as a vaccine adjuvant but also offer further insight into the understanding of the complex roles of GM-CSF

Three Essays on Corporate Finance

Archival abstract submitte

Three Essays on Corporate Finance

Archival abstract submitte