Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this p...Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.展开更多
Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdo...Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdomains. By modifying sucrose density gradient flotation centrifugation, which is used to isolate lipid microdomains, we were able to separate caveolae and noncaveolar lipid microdomains into two distinct fractions. The caveolar membranes are membrane vesicles of 100-nm diameter, enriched with caveolin-1 and flotillin-1. The noncaveolar lipid microdomains are amorphous membranes and most likely the coalescence of heterogeneous lipid rafts. They are depleted of caveo- lin-1 and are more enriched with cholesterol and sphingolipids than the caveolae. Many membrane proteins, such as insulin-like growth factor-1 receptor （membrane receptor）, aquaporin-1 （membrane transporter）, Thy-1 and N- cadherin （glycosylphosphatidylinositol-anchored membrane protein and membrane glycoprotein）, are specifically as- sociated with noncaveolar lipid microdomains, but not with caveolae. These results indicate that the lipid and protein compositions of caveolae differ from those of noncaveolar lipid microdomains. The difference in their protein compo- sitions implies that these two membrane microdomains may have different cellular functions.展开更多
While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow （CEF） around photosystem I （PSI） and cytochrome bef generates only ATP. CEF is thus essential to balance the supply of AT...While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow （CEF） around photosystem I （PSI） and cytochrome bef generates only ATP. CEF is thus essential to balance the supply of ATP and NADPH for carbon fixation; however, it remains unclear how the system tunes the relative levels of linear and cyclic flow. Here, we show that PETO, a transmembrane thylakoid phosphoprotein specific of green algae, contributes to the stimulation of CEF when cells are placed in anoxia. In oxic conditions, PETO co-fractionates with other thylakoid proteins involved in CEF （ANR1, PGRL1, FNR）. In PETO-knock- down strains, interactions between these CEF proteins are affected. Anoxia triggers a reorganization of the membrane, so that a subpopulation of PSi and cytochrome bsf now co-fractionates with the CEF effectors in sucrose gradients. The absence of PETO impairs this reorganization. Affinity purification identifies ANR1 as a major interactant of PETO. ANR1 contains two ANR domains, which are also found in the N-terminal region of NdhS, the ferredoxin-binding subunit of the plant ferredoxin-plastoquinone oxidoreductase （NDH）. We propose that the ANR domain was co-opted by two unrelated CEF systems （PGR and NDH）, possibly as a sensor of the redox state of the membrane.展开更多
The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, str...The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as pro- tein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly com- pacted than that in the exponential phase. In addition to factor for inversion stimulation （Fis）, histone-like nucleoid structuring protein （H-NS）, heat-unstable nucleoid protein （HU） and integration host factor （IHF） here we have identified, three new can- didates of E. coli nucleoid, namely DNA-binding protein from starved cells （Dps）, host factor for phage QJ3 （Hfq） and sup- pressor of taC phenotype A （StpA）. Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.展开更多
文摘Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.
文摘Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdomains. By modifying sucrose density gradient flotation centrifugation, which is used to isolate lipid microdomains, we were able to separate caveolae and noncaveolar lipid microdomains into two distinct fractions. The caveolar membranes are membrane vesicles of 100-nm diameter, enriched with caveolin-1 and flotillin-1. The noncaveolar lipid microdomains are amorphous membranes and most likely the coalescence of heterogeneous lipid rafts. They are depleted of caveo- lin-1 and are more enriched with cholesterol and sphingolipids than the caveolae. Many membrane proteins, such as insulin-like growth factor-1 receptor （membrane receptor）, aquaporin-1 （membrane transporter）, Thy-1 and N- cadherin （glycosylphosphatidylinositol-anchored membrane protein and membrane glycoprotein）, are specifically as- sociated with noncaveolar lipid microdomains, but not with caveolae. These results indicate that the lipid and protein compositions of caveolae differ from those of noncaveolar lipid microdomains. The difference in their protein compo- sitions implies that these two membrane microdomains may have different cellular functions.
文摘While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow （CEF） around photosystem I （PSI） and cytochrome bef generates only ATP. CEF is thus essential to balance the supply of ATP and NADPH for carbon fixation; however, it remains unclear how the system tunes the relative levels of linear and cyclic flow. Here, we show that PETO, a transmembrane thylakoid phosphoprotein specific of green algae, contributes to the stimulation of CEF when cells are placed in anoxia. In oxic conditions, PETO co-fractionates with other thylakoid proteins involved in CEF （ANR1, PGRL1, FNR）. In PETO-knock- down strains, interactions between these CEF proteins are affected. Anoxia triggers a reorganization of the membrane, so that a subpopulation of PSi and cytochrome bsf now co-fractionates with the CEF effectors in sucrose gradients. The absence of PETO impairs this reorganization. Affinity purification identifies ANR1 as a major interactant of PETO. ANR1 contains two ANR domains, which are also found in the N-terminal region of NdhS, the ferredoxin-binding subunit of the plant ferredoxin-plastoquinone oxidoreductase （NDH）. We propose that the ANR domain was co-opted by two unrelated CEF systems （PGR and NDH）, possibly as a sensor of the redox state of the membrane.
基金supported by Grants-in-Aid from the Ministry of Education,Science and Culture of Japan,and Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation
文摘The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as pro- tein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly com- pacted than that in the exponential phase. In addition to factor for inversion stimulation （Fis）, histone-like nucleoid structuring protein （H-NS）, heat-unstable nucleoid protein （HU） and integration host factor （IHF） here we have identified, three new can- didates of E. coli nucleoid, namely DNA-binding protein from starved cells （Dps）, host factor for phage QJ3 （Hfq） and sup- pressor of taC phenotype A （StpA）. Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.
