Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can direct...Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.展开更多
The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps3...The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins （SNXs）. Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2＇s dimerization partners, SNX6, interacts with the p150Gued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50Gued interaction. Disruption of the SNX6-pl50Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.展开更多
Aim To determine the effects of glucose on APD, I_(K1) , I_K , I_(Ca-L), ofventricular myocytes in guinea pigs, Methods Whole-cell patch-clamp technique was used to record thechanged action potential ionic current ind...Aim To determine the effects of glucose on APD, I_(K1) , I_K , I_(Ca-L), ofventricular myocytes in guinea pigs, Methods Whole-cell patch-clamp technique was used to record thechanged action potential ionic current induced by glucose of single cell in guinea pig ventricularmyocytes, to compare the action of 0, 10 and 20 mmol·L^(-1) glucoses on trans-membrane ioniccurrent. Results (1) Compared with 10 mmol·L^(-1) glucose concentrations, 0 and 20 mmol·L^(-1)glucose both shortened APD of ventricular myocytes ( P < 0.05). (2) The inward components ofI_(K1) density were maximal when the glucose concentration was at 10 mmol·L^(-1) . Normalized Ⅰ -Ⅴ relationships showed that both 0 and 20 mmol·L^(-1) glucose produced a left-shift of Ⅰ - Ⅴcurve. The reverse potential changed from - 72.4 mV to - 64.6 mV. (3) Compared with 10 mmol·L^(-1),both 0 and 20 mmol·L^(-1) glucose markedly increased the I_(Ca-L) amplitude and density. TheI_(Ca-L) current density was ( - 8.035 +- 0.82) pA/pF ( n = 8) at a test potential of 10 mV when theglucose concentration was 10 mmol·L^(-1) . But its current density decreased to ( - 5.45 +- 0.67)pA/pF and ( - 6.50 +- 0.56) pA/pF when glucose concentrations were 0 and 20 mmol·L^(-1) ,respectively. (4) The current densities of I_K were (18.96+-2.86) pA/pF, (8.66 +-1.87) pA/pF, and(15.32 +- 3.12) pA/pF, at + 70 mV for 0, 10 and 20 mmol·L^(-1) glucoses, respectively. ConclusionGlucose in different concentrations has different effects on APD, I_(K1), I_K, and I_(Ca-L) ofsingle ventricular myocyte in guinea pigs. There are similar actions of 0 and 20 mmol· L^(-1)glucoses on the transmembrane ionic current of ventricular myocytes in guinea pigs.展开更多
One of the major obstacles facing the field of structural biology in the post genomic era is the inherent difficulty of analyzing the structure of membrane proteins under native conditions. The method of choice for st...One of the major obstacles facing the field of structural biology in the post genomic era is the inherent difficulty of analyzing the structure of membrane proteins under native conditions. The method of choice for studying such proteins is FTIR spectroscopy. Following the outbreaking of the severe acute respiratory syndrome （SARS） virus, in 2003, extensive work has been directed at elucidating the structure of the E transmembrane proteins of the SARS coronavirus. In this study, the secondary structure of the transmembrane a-helical bundles was analysised using the biophysical method site specific infrared dichroism （SSID）. Sixteen amino acids were isotopically labeled with （~3C=180） at different positions of the primary structure of the synthesized E protein CoV. The secondary structure was studied using Attenuated Total Internal Reflection （ATR） FTIR spectroscopy. Based on our findings, the presence of two possible H-bonding interactions between the carbonyl oxygen of two residues 26 and 31 （Phe and Leu） respectively with water molecules which may be trapped within the helix structure were postulatesed. These interactions may cause a change in this structure.展开更多
基金supported by the Biotechnology and Biological Sciences Research Council and the Cystic Fibrosis Trust.H Li was supported by EuroCareCF(LSHM-CT-2005-018932)while J-H Chen and Z Xu were supported by scholarships from the University of Bristol and ORS awards from Universities UK.
基金Acknowledgments This study was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China （Grants 30425029, 30421001, 90717001）. We greatly thank Prof Hong Ma （Penn. State University, USA） for critical reading and writing improvement and Prof Nam-Hai Chua （The Rockefeller University, USA） for helpful comments. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Prof Sheng Luan （University of California, Berkeley, USA） for providing the construct pATC940. We thank Prof Hong-Quan Yang （SIPPE, CAS） for providing LexA yeast two-hybrid system and Prof Zhi-Yong Wang （The Stanford University, USA） for providing the BRI1 antibody. We thank Mr Xiao-Shu Gao for the help on Confocal Laser Scanning Microscopy.
文摘Brassinosteroids （BRs） are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmemhrane receptor BRI1 can directly bind to brassinolide （BL）, and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 （MSBP1） could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBPI specifically interacts with the extraeellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.
基金We thank Yingfang Liu （Institute of Biophysics, Chinese Acad- emy of Sciences） for advice on PX domain structure and SNX6 mutations. We are particularly grateful to Yanmin Yang （Stanford University, USA） for insightful discussions and the Flag-MAP1B LC construct. We also thank Juan S Bonifacino （NIH, USA） for the rabbit anti-CI-MPR antibody, Hiroyoshi Ariga （Hokkaido University, Japan） for Flag- and HA-tagged human SNX6 overexpression constructs, and Li Yu （Tsinghua University, China） for the YFP-EEA1 expression construct. We thank Chonglin Yang （Institute of Genetics and Developmental Biology, Chinese Academy of Sciences）, Dahua Chen （Institute of Zoology, Chinese Academy of Sciences） and Li Yu for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China （30770675） and Chinese Academy of Sciences （KSCX1-YW-R-37）. J-J Liu is supported by the CAS 100-Tal- ents Program.
文摘The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins （SNXs）. Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2＇s dimerization partners, SNX6, interacts with the p150Gued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50Gued interaction. Disruption of the SNX6-pl50Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.
文摘Aim To determine the effects of glucose on APD, I_(K1) , I_K , I_(Ca-L), ofventricular myocytes in guinea pigs, Methods Whole-cell patch-clamp technique was used to record thechanged action potential ionic current induced by glucose of single cell in guinea pig ventricularmyocytes, to compare the action of 0, 10 and 20 mmol·L^(-1) glucoses on trans-membrane ioniccurrent. Results (1) Compared with 10 mmol·L^(-1) glucose concentrations, 0 and 20 mmol·L^(-1)glucose both shortened APD of ventricular myocytes ( P < 0.05). (2) The inward components ofI_(K1) density were maximal when the glucose concentration was at 10 mmol·L^(-1) . Normalized Ⅰ -Ⅴ relationships showed that both 0 and 20 mmol·L^(-1) glucose produced a left-shift of Ⅰ - Ⅴcurve. The reverse potential changed from - 72.4 mV to - 64.6 mV. (3) Compared with 10 mmol·L^(-1),both 0 and 20 mmol·L^(-1) glucose markedly increased the I_(Ca-L) amplitude and density. TheI_(Ca-L) current density was ( - 8.035 +- 0.82) pA/pF ( n = 8) at a test potential of 10 mV when theglucose concentration was 10 mmol·L^(-1) . But its current density decreased to ( - 5.45 +- 0.67)pA/pF and ( - 6.50 +- 0.56) pA/pF when glucose concentrations were 0 and 20 mmol·L^(-1) ,respectively. (4) The current densities of I_K were (18.96+-2.86) pA/pF, (8.66 +-1.87) pA/pF, and(15.32 +- 3.12) pA/pF, at + 70 mV for 0, 10 and 20 mmol·L^(-1) glucoses, respectively. ConclusionGlucose in different concentrations has different effects on APD, I_(K1), I_K, and I_(Ca-L) ofsingle ventricular myocyte in guinea pigs. There are similar actions of 0 and 20 mmol· L^(-1)glucoses on the transmembrane ionic current of ventricular myocytes in guinea pigs.
文摘One of the major obstacles facing the field of structural biology in the post genomic era is the inherent difficulty of analyzing the structure of membrane proteins under native conditions. The method of choice for studying such proteins is FTIR spectroscopy. Following the outbreaking of the severe acute respiratory syndrome （SARS） virus, in 2003, extensive work has been directed at elucidating the structure of the E transmembrane proteins of the SARS coronavirus. In this study, the secondary structure of the transmembrane a-helical bundles was analysised using the biophysical method site specific infrared dichroism （SSID）. Sixteen amino acids were isotopically labeled with （~3C=180） at different positions of the primary structure of the synthesized E protein CoV. The secondary structure was studied using Attenuated Total Internal Reflection （ATR） FTIR spectroscopy. Based on our findings, the presence of two possible H-bonding interactions between the carbonyl oxygen of two residues 26 and 31 （Phe and Leu） respectively with water molecules which may be trapped within the helix structure were postulatesed. These interactions may cause a change in this structure.
