Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in...Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.展开更多
Dendrites are specialized neuronal compartments that sense, integrate and transfer information in the neural network. Their development is tightly controlled and abnormal dendrite morphogenesis is strongly linked to n...Dendrites are specialized neuronal compartments that sense, integrate and transfer information in the neural network. Their development is tightly controlled and abnormal dendrite morphogenesis is strongly linked to neurological disorders. While dendritic morphology ranges from relatively simple to extremely complex for a specified neuron, either requires a functional secretory pathway to continually replenish proteins and lipids to meet dendritic growth demands. The Golgi apparatus occupies the center of the secretory pathway and is regulating posttranslational modifications, sorting, transport, and signal transduction, as well as acting as a non-centrosomal microtubule organization center. The neuronal Golgi apparatus shares common features with Golgi in other eukaryotic cell types but also forms distinct structures known as Golgi outposts that specifically localize in dendrites. However, the organization and function of Golgi in dendrite development and its impact on neurological disorders is just emerging and so far lacks a systematic summary. We describe the organization of the Golgi apparatus in neurons, review the current understanding of Golgi function in dendritic morphogenesis, and discuss the current challenges and future directions.展开更多
Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too ...Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.展开更多
Poly(ADP-ribosyl)ation(PARylation)is a posttranslational modification reversibly catalyzed by poly(ADP-ribose)polymerases(PARPs)and poly(ADP-ribose)glycohydrolases(PARGs)and plays a key role in multi-ple cellular proc...Poly(ADP-ribosyl)ation(PARylation)is a posttranslational modification reversibly catalyzed by poly(ADP-ribose)polymerases(PARPs)and poly(ADP-ribose)glycohydrolases(PARGs)and plays a key role in multi-ple cellular processes.The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes.Here we show that Arabidopsis UBC13A and UBC13B,the major drivers of lysine 63(K63)-linked polyubiquitination,directly interact with PARPs/PARGs.Activation of pathogen-associated molecular pattern(PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13.Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related(PR)proteins but decreased accu-mulation of secreted PR proteins.Protein disulfide-isomerases(PDIs),essential components of endo-plasmic reticulum quality control(ERQC)that ensure proper folding and maturation of proteins destined for secretion,complex with PARPs/PARGs and are PARylated upon PAMP perception.Significantly,PARylation of UBC13 regulates K63-linked ubiquitination of PDIs,which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion.Taken together,these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.展开更多
Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular b...Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.展开更多
The regulatory mechanisms of cytoplasmic Ca2＋ after myocardial infarction-induced Ca2＋ overload involve secretory pathway Ca2＋-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi ...The regulatory mechanisms of cytoplasmic Ca2＋ after myocardial infarction-induced Ca2＋ overload involve secretory pathway Ca2＋-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca2＋ overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca2＋ concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca2＋ during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca2＋ showed an opposite effect. The expression of Golgi-specific secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was similar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca2＋-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca2＋-ATPase 1 plays an essential role in cytosolic calcium regulation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca2＋-ATPase 1 can be an important neuroprotective target of ischemic stroke.展开更多
基金This work is supported by the National Natural Science Foundation of China （30360061）Natural Science Foundation of Yunnan Province of China （1999C0008Z） National 863 Program of China （2003AA211020）.
文摘Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.
基金supported by grants from the National Natural Science Foundation of China(No.32000690 to CH)the Key-Area Research and Development Program of Guangdong Province,China(No.2019B030335001 to CH)+4 种基金the National Social Science Foundation of China(No.20&ZD296 to CH)the Deutsche Forschungsgemeinschaft(DFG.No.SO1337/4-1,SO1337/2-2,and SO1337/7-1 to PS)the DFG Heisenberg program(No.SO1337/6-1 to PS)ERA-NET NEURON(BMBFNo.01EW1910 and 01EW2108 to PS).
文摘Dendrites are specialized neuronal compartments that sense, integrate and transfer information in the neural network. Their development is tightly controlled and abnormal dendrite morphogenesis is strongly linked to neurological disorders. While dendritic morphology ranges from relatively simple to extremely complex for a specified neuron, either requires a functional secretory pathway to continually replenish proteins and lipids to meet dendritic growth demands. The Golgi apparatus occupies the center of the secretory pathway and is regulating posttranslational modifications, sorting, transport, and signal transduction, as well as acting as a non-centrosomal microtubule organization center. The neuronal Golgi apparatus shares common features with Golgi in other eukaryotic cell types but also forms distinct structures known as Golgi outposts that specifically localize in dendrites. However, the organization and function of Golgi in dendrite development and its impact on neurological disorders is just emerging and so far lacks a systematic summary. We describe the organization of the Golgi apparatus in neurons, review the current understanding of Golgi function in dendritic morphogenesis, and discuss the current challenges and future directions.
基金funded by grants from the Natural Science Foundation of China (Nos.31771600 and 31750410689)Tsinghua Initiative Program (No.20131089281)a 1000 Talents award,all to J.C.P.-P
文摘Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
基金supported by a start-up fund from Texas A&M AgriLife Research to J.S.a grant from the National Science Foundation(IOS-1951094)to P.H.and J.S.
文摘Poly(ADP-ribosyl)ation(PARylation)is a posttranslational modification reversibly catalyzed by poly(ADP-ribose)polymerases(PARPs)and poly(ADP-ribose)glycohydrolases(PARGs)and plays a key role in multi-ple cellular processes.The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes.Here we show that Arabidopsis UBC13A and UBC13B,the major drivers of lysine 63(K63)-linked polyubiquitination,directly interact with PARPs/PARGs.Activation of pathogen-associated molecular pattern(PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13.Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related(PR)proteins but decreased accu-mulation of secreted PR proteins.Protein disulfide-isomerases(PDIs),essential components of endo-plasmic reticulum quality control(ERQC)that ensure proper folding and maturation of proteins destined for secretion,complex with PARPs/PARGs and are PARylated upon PAMP perception.Significantly,PARylation of UBC13 regulates K63-linked ubiquitination of PDIs,which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion.Taken together,these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.
基金the National Natural Science Foundation of China(Grant No.30470863)
文摘Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.
基金supported by the National Natural Science Foundation of China,No.81171239
文摘The regulatory mechanisms of cytoplasmic Ca2＋ after myocardial infarction-induced Ca2＋ overload involve secretory pathway Ca2＋-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca2＋ overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca2＋ concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca2＋ during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca2＋ showed an opposite effect. The expression of Golgi-specific secretory pathway Ca2＋-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was similar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca2＋-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca2＋-ATPase 1 plays an essential role in cytosolic calcium regulation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca2＋-ATPase 1 can be an important neuroprotective target of ischemic stroke.
