We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in h... more We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in human red blood cell membranes and in Clone 9 cells. We show here that in a mixed population of stably transfected cells, overexpression of either murine or human stomatin resulted in 35–50% reduction in the basal rate of glucose transport. Moreover, there was a correlation between increased expression of stomatin and depression in the rate of glucose transport. In two clones chosen for further study, the ∼10% and ∼70% reduction in basal rate of glucose transport was associated with increases in stomatin mRNA and protein expression without a detectable change in GLUT-1 content in plasma membranes of either clone. In the clone overexpressing high levels of stomatin, immunoprecipitated GLUT-1 was associated with a large amount of stomatin as a coimmunoprecipitant. Employing extracts of cells overexpressing human stomatin, we found that stomatin bound to the glutathione- S-transferase (GST) ...
Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton ac... more Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/cho...
Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely e... more Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocy...
Advances in Planar Lipid Bilayers and Liposomes, 2008
... All rights reserved. Permissions & Reprints. Chapter Three Insights in the Organization a... more ... All rights reserved. Permissions & Reprints. Chapter Three Insights in the Organization and Dynamics of Erythrocyte Lipid Rafts. Ulrich Salzer Corresponding Author Contact Information , a , E-mail The Corresponding Author , Ursula Hunger a and Rainer Prohaska a. ...
Cytosolic Ca11 induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic f... more Cytosolic Ca11 induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glyco- syl phosphatidylinositol-linked proteins, free of cytoskeletal components, and de- pleted of the major transmembrane pro- teins. Both types of
ABSTRACT When human erythrocytes are treated with Ca2+ and ionophore A23187, two kinds of vesicle... more ABSTRACT When human erythrocytes are treated with Ca2+ and ionophore A23187, two kinds of vesicles are shed from the surface, socalled microvesicles and nanovesicles. These vesicles were purified by differential centrifugation and their sizes and structures were determined by dynamic-force microscopy under physiological conditions. Microvesicles (Fig. 1a) and nanovesicles (Fig. 1b) were significantly different in size. Their size distributions showed maxima at 179 nm and 81 nm diameter, respectively. Higher magnification amplitude images resolved a patchy fine structure of the microvesicle membrane (Fig. 1c). Because microvesicles are free of cytoskeletal components, this fine structure apparently reflects the inherent domain organization of the vesicular membrane. Some of the microvesicles are associated with flat vesicular membrane domains (Fig. 1c), possibly indicating a domain segregation. Biochemical analysis revealed that microvesicles contain lipid rafts enriched in glycosyl phosphatidylinositol (GPI)-linked proteins, the lipid raft protein stomatin, and two cytosolic proteins, synexin and sorcin, which translocate to the membrane upon Ca2+ binding. Nanovesicles also contain lipid rafts, with synexin and sorcin being the most abundant proteins in the presence of Ca2+. Interestingly, whereas stomatin is specifically present in the microvesicular rafts, synexin and sorcin are present in microvesicular and nanovesicular rafts, the lipid raft proteins flotillin-1 and -2 are not found in the vesicles but remain in the red cell membrane. These data indicate the coexistence of different types of lipid rafts in the membrane and their segregation upon treatment with Ca2+. Synexin is suggested to be the driving force for the release of vesicles from erythrocytes.
Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was... more Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.
We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in h... more We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in human red blood cell membranes and in Clone 9 cells. We show here that in a mixed population of stably transfected cells, overexpression of either murine or human stomatin resulted in 35–50% reduction in the basal rate of glucose transport. Moreover, there was a correlation between increased expression of stomatin and depression in the rate of glucose transport. In two clones chosen for further study, the ∼10% and ∼70% reduction in basal rate of glucose transport was associated with increases in stomatin mRNA and protein expression without a detectable change in GLUT-1 content in plasma membranes of either clone. In the clone overexpressing high levels of stomatin, immunoprecipitated GLUT-1 was associated with a large amount of stomatin as a coimmunoprecipitant. Employing extracts of cells overexpressing human stomatin, we found that stomatin bound to the glutathione- S-transferase (GST) ...
Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton ac... more Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/cho...
Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely e... more Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocy...
Advances in Planar Lipid Bilayers and Liposomes, 2008
... All rights reserved. Permissions & Reprints. Chapter Three Insights in the Organization a... more ... All rights reserved. Permissions & Reprints. Chapter Three Insights in the Organization and Dynamics of Erythrocyte Lipid Rafts. Ulrich Salzer Corresponding Author Contact Information , a , E-mail The Corresponding Author , Ursula Hunger a and Rainer Prohaska a. ...
Cytosolic Ca11 induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic f... more Cytosolic Ca11 induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glyco- syl phosphatidylinositol-linked proteins, free of cytoskeletal components, and de- pleted of the major transmembrane pro- teins. Both types of
ABSTRACT When human erythrocytes are treated with Ca2+ and ionophore A23187, two kinds of vesicle... more ABSTRACT When human erythrocytes are treated with Ca2+ and ionophore A23187, two kinds of vesicles are shed from the surface, socalled microvesicles and nanovesicles. These vesicles were purified by differential centrifugation and their sizes and structures were determined by dynamic-force microscopy under physiological conditions. Microvesicles (Fig. 1a) and nanovesicles (Fig. 1b) were significantly different in size. Their size distributions showed maxima at 179 nm and 81 nm diameter, respectively. Higher magnification amplitude images resolved a patchy fine structure of the microvesicle membrane (Fig. 1c). Because microvesicles are free of cytoskeletal components, this fine structure apparently reflects the inherent domain organization of the vesicular membrane. Some of the microvesicles are associated with flat vesicular membrane domains (Fig. 1c), possibly indicating a domain segregation. Biochemical analysis revealed that microvesicles contain lipid rafts enriched in glycosyl phosphatidylinositol (GPI)-linked proteins, the lipid raft protein stomatin, and two cytosolic proteins, synexin and sorcin, which translocate to the membrane upon Ca2+ binding. Nanovesicles also contain lipid rafts, with synexin and sorcin being the most abundant proteins in the presence of Ca2+. Interestingly, whereas stomatin is specifically present in the microvesicular rafts, synexin and sorcin are present in microvesicular and nanovesicular rafts, the lipid raft proteins flotillin-1 and -2 are not found in the vesicles but remain in the red cell membrane. These data indicate the coexistence of different types of lipid rafts in the membrane and their segregation upon treatment with Ca2+. Synexin is suggested to be the driving force for the release of vesicles from erythrocytes.
Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was... more Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.
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