Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

scholarly journals Antidiuretic hormone modulates membrane phosphoproteins in toad urinary bladder and retrieved water channel containing apical membrane vesicles.

1991 ◽  
Vol 1 (9) ◽  
pp. 1114-1122
Author(s):  
H W Harris
Keyword(s):  
Membrane Proteins ◽  
Urinary Bladder ◽  
Membrane Protein ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Granular Cells

Antidiuretic hormone (ADH) dramatically increases the water permeability of toad urinary bladder by insertion of unique highly selective water channels into the apical membranes of granular cells. Before ADH stimulation, water channels are stored in high concentrations in the limiting membranes of large cytoplasmic vesicles called aggrephores. ADH stimulation causes aggrephore fusion with the granular cell apical membrane and increases water permeability. Transepithelial osmotic water flow causes a rapid attenuation of the ADH-elicited increase in water permeability through a process called flux inhibition. Flux inhibition is due to retrieval of ADH water channels by apical membrane endocytosis. When phosphoproteins of intact bladders are labeled with (32P)orthophosphate, the 32P content of 34-, 28-, and 17-kDa proteins is increased by ADH stimulation. When flux inhibition occurs, the 32P-labelling of a 15.5-kDa protein is reduced to approximately one half its original value (Konieczkowski M, Rudolph SA, J Pharmacol Exp Ther 1985;234:515). These observations have been confirmed, and these studies have been extended, by using a combination of subcellular fractionation and membrane protein chemistry techniques. All four of these phosphoproteins are present in membrane fractions of granular cells. Analysis of membrane proteins by a combination of Triton X-114 partitioning and an alkaline stripping technique reveals that the 28- and 17-kDa species are integral membrane proteins of unknown function. In contrast, the 32P-labeled 15.5-kDa protein is a peripheral membrane protein. It is attached to the cytoplasmic (outer) surface of highly water-permeable vesicles retrieved during flux inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane

1993 ◽  
Vol 265 (3) ◽  
pp. C822-C833 ◽  
Author(s):  
M. L. Zeidel ◽  
T. G. Hammond ◽  
J. B. Wade ◽  
J. Tucker ◽  
H. W. Harris
Keyword(s):  
Membrane Proteins ◽  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Water Channel ◽  
Fluid Phase ◽  
Water Channels ◽  
Endocytic Pathway ◽  
Electron Microscopic ◽  
Channel Proteins ◽  
Fluorescence Measurements

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

1996 ◽  
Vol 109 (6) ◽  
pp. 1215-1227 ◽  
Author(s):  
I. Hemery ◽  
A.M. Durand-Schneider ◽  
G. Feldmann ◽  
J.P. Vaerman ◽  
M. Maurice
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Rat Hepatocytes ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Plasma Membrane Protein ◽  
Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

Vasopressin decreases immunogold labeling of apical actin in the toad bladder granular cell

1992 ◽  
Vol 263 (4) ◽  
pp. C908-C912 ◽  
Author(s):  
Y. Gao ◽  
N. Franki ◽  
F. Macaluso ◽  
R. M. Hays
Keyword(s):  
Actin Cytoskeleton ◽  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Water Channel ◽  
Granular Cell ◽  
Immunogold Labeling ◽  
Toad Bladder ◽  
Apical Region ◽  
Electron Microscopic ◽  
Relative Extent

Studies with the confocal microscope have shown that arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the toad bladder granular cell. However, the resolution of the fluorescence microscope is not great enough to reveal the exact pattern of depolymerization or the relative extent to which microvillar and subapical membrane actin pools contribute to overall depolymerization. We have developed an electron microscopic immunogold method that shows a significant decrease in immunogold labeling of actin in the region just below the apical membrane, with the decrease most pronounced in regions adjacent to the microvilli. There was no significant change of immunogold labeling within the microvilli themselves. Our studies show a reorganization of the actin cytoskeleton in the region of the granular cell, where water channel-carrying vesicles are positioned and fuse in response to AVP.

scholarly journals Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies

2003 ◽  
Vol 2003 (4) ◽  
pp. 249-255 ◽  
Author(s):  
M. Walid Qoronfleh ◽  
Betsy Benton ◽  
Ray Ignacio ◽  
Barbara Kaboord
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Extraction ◽  
Yeast Cells ◽  
Protein Markers ◽  
Two Phase ◽  
Western Blots ◽  
Selective Enrichment ◽  
Phase Partitioning ◽  
Hydrophobic Fraction

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.

Water channel-carrying vesicles in the rat IMCD contain cellubrevin

1995 ◽  
Vol 269 (3) ◽  
pp. C797-C801 ◽  
Author(s):  
N. Franki ◽  
F. Macaluso ◽  
W. Schubert ◽  
L. Gunther ◽  
R. M. Hays
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunogold Electron Microscopy ◽  
Cyclic Process ◽  
Docking Proteins ◽  
Medullary Collecting Duct

Antidiuretic hormone (arginine vasopressin) induces a cyclic process of docking, fusion, and endocytosis of water channel-containing vesicles in the collecting duct. There is now evidence that docking and endocytosis are mediated by an array of proteins associated with vesicles and target membranes. In recent studies, we have shown that cellubrevin, a member of the vesicle-associated membrane protein family, as well as other docking proteins, are expressed in the rat inner medullary collecting duct. We now show by immunogold electron microscopy that cellubrevin is present on vesicles containing water channels, that it is associated with both coated and uncoated vesicles, and that it is present on the apical membrane. Cellubrevin, therefore, is in a position to mediate one or more steps in arginine vasopressin-induced water channel cycling.

scholarly journals Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JM Gerrard ◽  
D Lint ◽  
PJ Sims ◽  
T Wiedmer ◽  
RD Fugate ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein S ◽  
Dense Granule ◽  
Western Blots ◽  
Dense Granules ◽  
Granule Membrane ◽  
Thrombin Activation ◽  
Hermansky Pudlak Syndrome ◽  
Slot Blot Analysis

Abstract Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

scholarly journals YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein

2013 ◽  
Vol 288 (23) ◽  
pp. 16295-16307 ◽  
Author(s):  
Ilie Sachelaru ◽  
Narcis Adrian Petriman ◽  
Renuka Kudva ◽  
Patrick Kuhn ◽  
Thomas Welte ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Transmembrane Domains ◽  
Cross Linking ◽  
Lipid Phase ◽  
Lipid Transfer ◽  
Lateral Gate

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.

scholarly journals Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JM Gerrard ◽  
D Lint ◽  
PJ Sims ◽  
T Wiedmer ◽  
RD Fugate ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein S ◽  
Dense Granule ◽  
Western Blots ◽  
Dense Granules ◽  
Granule Membrane ◽  
Thrombin Activation ◽  
Hermansky Pudlak Syndrome ◽  
Slot Blot Analysis

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

scholarly journals Membrane protein-based biosensors

2018 ◽  
Vol 15 (141) ◽  
pp. 20170952 ◽  
Author(s):  
Nobuo Misawa ◽  
Toshihisa Osaki ◽  
Shoji Takeuchi
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Recent Progress ◽  
Biological Membranes ◽  
Cultured Cell ◽  
Sensor Device ◽  
Fundamental Information ◽  
Essential Components ◽  
Target Molecules

This review highlights recent development of biosensors that use the functions of membrane proteins. Membrane proteins are essential components of biological membranes and have a central role in detection of various environmental stimuli such as olfaction and gustation. A number of studies have attempted for development of biosensors using the sensing property of these membrane proteins. Their specificity to target molecules is particularly attractive as it is significantly superior to that of traditional human-made sensors. In this review, we classified the membrane protein-based biosensors into two platforms: the lipid bilayer-based platform and the cell-based platform. On lipid bilayer platforms, the membrane proteins are embedded in a lipid bilayer that bridges between the protein and a sensor device. On cell-based platforms, the membrane proteins are expressed in a cultured cell, which is then integrated in a sensor device. For both platforms we introduce the fundamental information and the recent progress in the development of the biosensors, and remark on the outlook for practical biosensing applications.

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