The accessory proteins(3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins(PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus(SARS-Co V) genom...The accessory proteins(3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins(PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus(SARS-Co V) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP(enhanced yellow fluorescent protein) bimolecular fluorescence complementation(BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid(Y2H)system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions.展开更多
[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further ...[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.展开更多
An increasing body of evidence shows that the lipid droplet,a neutral lipid storage organelle,plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria.However,the cellular func...An increasing body of evidence shows that the lipid droplet,a neutral lipid storage organelle,plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria.However,the cellular functions and molecular mechanisms of the interaction remain ambiguous.Here we present data from transmission electron microscopy,fluorescence imaging,and reconstitution assays,demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro.Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae,we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes.The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75%of the interactions detected.Interestingly,interactions between 3 pairs of lipid metabolic enzymes were detected.Collectively,these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes,and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.展开更多
Genome-wide physical protein±protein interaction(PPI)mapping remains a major challenge for current technologies.Here,we reported a high-efficiency BiFC-seq method,yeastenhanced green fluorescent protein-based bim...Genome-wide physical protein±protein interaction(PPI)mapping remains a major challenge for current technologies.Here,we reported a high-efficiency BiFC-seq method,yeastenhanced green fluorescent protein-based bimolecular fluorescence complementation(y EGFPBiFC)coupled with next-generation DNA sequencing,for interactome mapping.We first applied y EGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus.Two-thirds(9/14)of known interactions of EBOV were recaptured,and five novel interactions were discovered.Next,we used the BiFC-seq method to map the interactome of the tumor protein p53.We identified 97 interactors of p53,more than three-quarters of which were novel.Furthermore,in a more complex background,we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins.These results show that BiFC-seq is a highly sensitive,rapid,and economical method for genome-wide interactome mapping.展开更多
基金supported by National Natural Science Foundation of China (No. 81072673)
文摘The accessory proteins(3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins(PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus(SARS-Co V) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP(enhanced yellow fluorescent protein) bimolecular fluorescence complementation(BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid(Y2H)system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions.
基金Supported by National Natural Science Foundation of China(30870109)~~
文摘[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.
基金supported by grants from the National Basic Research Program of China(Grant Nos.2009CB919000 and 2010CB833703)the National Natural Science Foundation of China(Grant Nos.30871229 and 30971431)the 21C Frontier Functional Proteomics Project(FPR08A1-060)funded by the Ministry of Education,Science and Technology,Republic of Korea.
文摘An increasing body of evidence shows that the lipid droplet,a neutral lipid storage organelle,plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria.However,the cellular functions and molecular mechanisms of the interaction remain ambiguous.Here we present data from transmission electron microscopy,fluorescence imaging,and reconstitution assays,demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro.Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae,we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes.The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75%of the interactions detected.Interestingly,interactions between 3 pairs of lipid metabolic enzymes were detected.Collectively,these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes,and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.
基金supported by grants from the National Key R&D Program of China(Grant No.2017YFA0505700)the National Key Lab of Proteomics of China(Grant Nos.SKLP-K201805,SKLP-K201804,and SKLP-Y201703)。
文摘Genome-wide physical protein±protein interaction(PPI)mapping remains a major challenge for current technologies.Here,we reported a high-efficiency BiFC-seq method,yeastenhanced green fluorescent protein-based bimolecular fluorescence complementation(y EGFPBiFC)coupled with next-generation DNA sequencing,for interactome mapping.We first applied y EGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus.Two-thirds(9/14)of known interactions of EBOV were recaptured,and five novel interactions were discovered.Next,we used the BiFC-seq method to map the interactome of the tumor protein p53.We identified 97 interactors of p53,more than three-quarters of which were novel.Furthermore,in a more complex background,we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins.These results show that BiFC-seq is a highly sensitive,rapid,and economical method for genome-wide interactome mapping.
