为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对...为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对,转染后筛选获得Mon1a基因敲除的HEK293T稳定细胞系。进一步从细胞增殖能力、迁移能力以及小鼠皮下成瘤能力等方面研究了Mon1a缺失对HEK293T细胞的影响。结果表明,Mon1a在细胞增殖、迁移和成瘤等过程中起到重要作用。展开更多
Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome,also named the vacuole in plants.However,the molecular mechanisms unde...Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome,also named the vacuole in plants.However,the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear.Here,we report the functional characterization of a rice(Oryza sativa)mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells.Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles.The mutant harbors a loss-of-function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1(MON1).Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY-RELATED 8a in maintaining normal growth and development.In addition,the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles.Furthermore,quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport,thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles.Together,our findings establish a molecular link between autophagy and vacuolar protein transport,and offer insights into the dual functions of the MON1–CCZ1(CAFFEINE ZINC SENSITIVITY1)complex in plants.展开更多
Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a gua...Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.展开更多
文摘为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对,转染后筛选获得Mon1a基因敲除的HEK293T稳定细胞系。进一步从细胞增殖能力、迁移能力以及小鼠皮下成瘤能力等方面研究了Mon1a缺失对HEK293T细胞的影响。结果表明,Mon1a在细胞增殖、迁移和成瘤等过程中起到重要作用。
基金supported by grants from the National Key R&D Program of China(2021YFF1000200)Innovation Program of Chinese Academy of Agricultural Sciences,International Science&Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-ZDRW202109)+2 种基金National Natural Science Foundation of China(31830064 and 32172085)supported by the Central Public-Interest Scientific Institution Basal Research Fund,China(Y2021YJ18)Jiangsu Nanjing Rice Germplasm Resources National Field Observation and Research Station。
文摘Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome,also named the vacuole in plants.However,the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear.Here,we report the functional characterization of a rice(Oryza sativa)mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells.Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles.The mutant harbors a loss-of-function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1(MON1).Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY-RELATED 8a in maintaining normal growth and development.In addition,the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles.Furthermore,quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport,thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles.Together,our findings establish a molecular link between autophagy and vacuolar protein transport,and offer insights into the dual functions of the MON1–CCZ1(CAFFEINE ZINC SENSITIVITY1)complex in plants.
基金supported by the grants from the National Natural Science Foundation of China(32201025 to D.T.,32071214 to S.Q.,32470738 to S.Q.,and 32471311 to D.T.)。
文摘Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.
