The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, al...The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.展开更多
As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear tra...As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.展开更多
Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a p...Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.展开更多
AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis.
Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, pos...Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer （TOC） and inner envelope （TOC）. The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semiconserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit （SStp） and ferredoxin （Fdtp）. Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations （-85） in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.展开更多
Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components o...Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components of the intermembrane space （IMS）. That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.展开更多
AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression...AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NSSA-expressed and non-HCV NSSA-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.RESULTS： Expression of HCV NS5A was found in the cytoplasm of pCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells, STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction, STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to pRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.CONCLUSION： HCV NSSA expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.展开更多
Thousands of nuclear-encoded proteins are transported into chloroplasts through the TOC–TIC translocon that spans the chloroplast envelope membranes.A motor complex pulls the translocated proteins out of the TOC–TIC...Thousands of nuclear-encoded proteins are transported into chloroplasts through the TOC–TIC translocon that spans the chloroplast envelope membranes.A motor complex pulls the translocated proteins out of the TOC–TIC complex into the chloroplast stroma by hydrolyzing ATP.The Orf2971–FtsHi complex has been suggested to serve as the ATP-hydrolyzing motor in Chlamydomonas reinhardtii,but little is known about its architecture and assembly.Here,we report the 3.2-Åresolution structure of the Chlamydomonas Orf2971–FtsHi complex.The 20-subunit complex spans the chloroplast inner envelope,with two bulky modules protruding into the intermembrane space and stromal matrix.Six subunits form a hetero-hexamer that potentially provides the pulling force through ATP hydrolysis.The remaining subunits,including potential enzymes/chaperones,likely facilitate the complex assembly and regulate its proper function.Taken together,our results provide the structural foundation for a mechanistic understanding of chloroplast protein translocation.展开更多
Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively...Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.展开更多
Unconventional protein export/secretion(UPE/UPS),in contrast to the classical ER-Golgi-dependent export/secretion of proteins with a leader sequence(signal peptide),employs multiple means to release leaderless cargoes...Unconventional protein export/secretion(UPE/UPS),in contrast to the classical ER-Golgi-dependent export/secretion of proteins with a leader sequence(signal peptide),employs multiple means to release leaderless cargoes(and in some special cases,cargoes with a leader sequence)to the extracellular space.By far,two major types of UPE have been classified,vesicle-independent UPE and vesicle-dependent UPE.In the former,UPE cargoes can directly translocate across the plasma membrane from the cytoplasm without the assistance of a vesicle carrier.In the latter,UPE cargoes translocate into the lumen of a vesicle which then delivers them out of the cell through membrane trafficking.Both types of UPE require multiple unconventional solutions to complete secretion.Here,we briefly discuss the multiple strategies for a UPE cargo release,focusing on two key steps of leaderless cargoes release in UPE:protein translocation and membrane trafficking.展开更多
文摘The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.
基金supported by the National Key Research and Development Program of China (2017YFC1001303)International Cooperation Project of China and Canada NSFC (81661128010)+2 种基金National Natural Science Foundation of China (31471405, 81671456, 81671412)the National Key Basic Research Program (2013CB967404) the Doctoral Innovation Fund of School of Medicine, Shanghai Jiao Tong University (BXJ201640)
文摘As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.
文摘Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.
基金Supported by National Natural Science Foundation of China,No.81072040
文摘AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis.
文摘Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer （TOC） and inner envelope （TOC）. The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semiconserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit （SStp） and ferredoxin （Fdtp）. Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations （-85） in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.
文摘Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane （TOC and TIC）. It was suggested that both machineries are interconnected by components of the intermembrane space （IMS）. That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.
基金Supported by National Natural Science Foundation of ChinaNo. 39670671, No. 30471531
文摘AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NSSA-expressed and non-HCV NSSA-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.RESULTS： Expression of HCV NS5A was found in the cytoplasm of pCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells, STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction, STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to pRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.CONCLUSION： HCV NSSA expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.
基金funded by the Strategic Priority Research Program of CAS(XDB37020101)the National Key R&D Program of China(2021YFA0910800)+3 种基金the National Natural Science Foundation of China(31930064)the Youth Innovation Promotion Association,Chinese Academy of Sciences(Y2022038)the Regional Joint Key Projects of the National Foundation of China(U22A20445)the Natural Science Foundation of Shandong Province(ZR2023ZD30).
文摘Thousands of nuclear-encoded proteins are transported into chloroplasts through the TOC–TIC translocon that spans the chloroplast envelope membranes.A motor complex pulls the translocated proteins out of the TOC–TIC complex into the chloroplast stroma by hydrolyzing ATP.The Orf2971–FtsHi complex has been suggested to serve as the ATP-hydrolyzing motor in Chlamydomonas reinhardtii,but little is known about its architecture and assembly.Here,we report the 3.2-Åresolution structure of the Chlamydomonas Orf2971–FtsHi complex.The 20-subunit complex spans the chloroplast inner envelope,with two bulky modules protruding into the intermembrane space and stromal matrix.Six subunits form a hetero-hexamer that potentially provides the pulling force through ATP hydrolysis.The remaining subunits,including potential enzymes/chaperones,likely facilitate the complex assembly and regulate its proper function.Taken together,our results provide the structural foundation for a mechanistic understanding of chloroplast protein translocation.
文摘Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.
基金supported by the National Natural Science Foundation of China(32130023,91854114,and 32061143009)the Ministry of Science and Technology of the People’s Republic of China(2019YFA0508602 and 2021YFA0804802)the Beijing Natural Science Foundation(JQ20028)。
文摘Unconventional protein export/secretion(UPE/UPS),in contrast to the classical ER-Golgi-dependent export/secretion of proteins with a leader sequence(signal peptide),employs multiple means to release leaderless cargoes(and in some special cases,cargoes with a leader sequence)to the extracellular space.By far,two major types of UPE have been classified,vesicle-independent UPE and vesicle-dependent UPE.In the former,UPE cargoes can directly translocate across the plasma membrane from the cytoplasm without the assistance of a vesicle carrier.In the latter,UPE cargoes translocate into the lumen of a vesicle which then delivers them out of the cell through membrane trafficking.Both types of UPE require multiple unconventional solutions to complete secretion.Here,we briefly discuss the multiple strategies for a UPE cargo release,focusing on two key steps of leaderless cargoes release in UPE:protein translocation and membrane trafficking.
