Distribution of Transmembrane Polypeptides in Freeze Fracture

Edwards, Harold H.; Mueller, Thomas; Morrison, Martin

doi:10.1126/science.424755

Cited by 57 publications

(36 citation statements)

References 33 publications

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“…Labeling of the P face by Con A-ferritin conjugates implies that band 3 component (the Con A binding transmembrane protein ; see references [4][5][6][7][8] generally partitions with the inner membrane half, a process that must involve dragging of its Con A binding heterosaccharides across the outer membrane half. These results are consistent with the biochemical analysis of half-membrane preparations (9) and, in addition, they indicate that Con A labeling of the E face, although minor, appears significant .…”

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confidence: 90%

“…Biochemical studies indicate the presence of glycophorin molecules and shorter sialoglycopeptides in preparations of exoplasmic membrane halves (9). These studies, however, were not designed to assess the degree of partition of glycophorin with the inner membrane half, nor could this partition be inferred from our initial fracture-label studies (1) showing the presence of cationized ferritin or colloidal iron on both P and E faces, as these markers cannot identify the molecular species labeled .…”

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Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Silva¹,

Torrisi²

1982

The Journal of Cell Biology

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Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin-associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces . Label is sparse over the protoplasmic faces . These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face . Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freezefracture . This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton .In freeze-fracture, splitting of the bilayer membrane continuum with differential partition of integral membrane proteins can be considered a process of structural dissection . Recently, we developed "fracture-label" techniques for the direct cytochemical investigation of the distribution and partition of integral and peripheral membrane components as indicated by the presence of chemical groups and lectin binding sites on the faces of freeze-fractured plasma and intracellular membranes . The results can be observed both in thin sections of freezefractured material ("thin section fracture-label") (1, 2) and in platinum-carbon replicas ofcritical-point-dried specimens after freeze-fracture ("critical point drying fracture-label") (3) .Initial application of thin-section fracture-label to the cytochemical investigation of the fracture faces of human erythrocyte membranes showed that concanavalin A (Con A) binding sites are preferentially labeled on the protoplasmic faces (1) . Labeling of the P face by Con A-ferritin conjugates implies that band 3 component (the Con A binding transmembrane protein ; see references 4-8) generally partitions with the inner membrane half, a process that must involve dragging of its Con A binding heterosaccharides across the outer membrane half. These results are consistent with the biochemical analysis of half-membrane preparations (9) and, in addition, they indicate that Con A labeling of the E face, although minor, appears significant .Much less is known about the partition of glycophorin . Biochemical studies indicate the presence of glycophorin molecules and shorter sialoglycopeptides in preparations of exoplasmic membrane halves (9). These studies, however, were not designed to assess the degree of partition of glycophorin with the inner membrane half, nor could this partition be inferred from our initial fracture-label studies (1) showing the presence of cationized ferritin or colloidal iron on both P and E faces, as these markers cannot identify the molecul...

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Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Silva¹,

Torrisi²

1982

The Journal of Cell Biology

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“…Since band 3 is the major component of intramembrane particles (43,44), we measured the size and number ofthese particles. Fig.…”

Section: Band 3 Content Of Density-separated Red Cells Since Thementioning

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Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

et al. 1994

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We describe a duplication of 10 nucleotides (2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis. (J. Clin. Invest. 1994. 93:121-130.) Key words: erythrocyte membrane band 3 protein * congenital hemolytic anemia * frameshift mutation -density gradient centrifugation * transmission electron microscopy

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“…Although splitting of the bilayer continuum by freeze-fracture (and, in consequence, the partition of peripheral membrane proteins) is established, less is known about the fracture behavior of integral membrane proteins (3,16)-in particular, their antigens and lectin binding sites at the surface (17). Most particles are generally associated with the inner membrane half (1, 2)-i. e., they are observed on the protoplasmic or P face (18).…”

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Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

Silva¹,

Parkison²,

Dwyer³

1981

Proc. Natl. Acad. Sci. U.S.A.

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A method-"fracture label"-is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the. inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragd from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new. anionic-sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed. by freeze-fracture of isolated cells and tissues.Freeze-fracture splits membranes along their hydrophobic interior, following the juncture ofend groups provided by a membrane continuum with bilayer organization (1-4). Although fracture of the bilayer produces smooth faces, in all biological membranes these faces are interrupted by particulate components ("membrane intercalated particles" or "intramembranous particles") and, in most, by subtler rugosities ["subparticles" (5)]. Combined use ofimmunochemical and cytochemical techniques with freeze-etching methods, and freeze-fracture observation of reconstituted membrane preparations, demonstrate that, in the systems used, the particles represent the sites ofintegral transmembrane proteins (6-12). Because of the qualitative similarity offreeze-fracture images produced by all biological membranes, the particles are assumed to represent local structural asymmetries in the fracture process provoked by integral membrane.proteins and, possibly, their-associated lipids (3, 4). In the erythrocyte membrane the particles represent the site of the two main integral transmembrane proteins (glycophorin and band III component) which bear AB(H) antigens, influenza virus receptors, wheat germ agglutinin and concanavalin A (Con A) binding sites, as well as anionic sites (8)(9)(10)(11)(13)(14)(15).Although splitting of the bilayer continuum by freeze-fracture (and, in consequence, the partition of peripheral membrane proteins) is established, less is known about the fracture behavior of integral membrane proteins (3, 16)-in particular, their antigens and lectin binding sites at the surface (17). Most particles are generally associated with the inner membrane half (1, 2)-i. e., they are observed on the protoplasmic or P face (18). This may be the result ofa stochastic process or reflect individual differences of fracture behavior of the components in each particle or both. On face P the particles represent, at least in part, portions of integral proteins located at the outer (exoplasmic, E) membrane half. It is unknown whether the particles contain hydrophilic surface groups ofintegral proteins dragged, during fracture, from the outer surface. We have labeled freezefractured erythrocytes and repor...

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Distribution of Transmembrane Polypeptides in Freeze Fracture

Cited by 57 publications

References 33 publications

Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

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