scholarly journals Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.

1987 ◽  
Vol 105 (3) ◽  
pp. 1241-1251 ◽  
Author(s):  
J R Bartles ◽  
H M Feracci ◽  
B Stieger ◽  
A L Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Rat Hepatocyte ◽  
Dipeptidylpeptidase Iv ◽  
Plasma Membrane Fraction ◽  
Apical Domain ◽  
Basolateral Plasma Membrane

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.

scholarly journals The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells

1992 ◽  
Vol 283 (2) ◽  
pp. 553-560 ◽  
Author(s):  
J A Ellis ◽  
M R Jackman ◽  
J P Luzio
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Fraction ◽  
Apical Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Basolateral Membrane ◽  
Subcellular Fractionation ◽  
Integral Membrane Proteins ◽  
Basolateral Plasma Membrane

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5′-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells

1991 ◽  
Vol 261 (3) ◽  
pp. C543-C549 ◽  
Author(s):  
J. Torchia ◽  
Y. Qu ◽  
J. Francis ◽  
D. J. Pon ◽  
A. K. Sen
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Intact Cell ◽  
Fold Increase ◽  
Salt Gland ◽  
Subcellular Fractionation ◽  
Cellular Protein ◽  
Isolated Cells ◽  
Plasma Membrane Fraction ◽  
Gland Cells

The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.

scholarly journals Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

1990 ◽  
Vol 1025 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Joseph W. Francis ◽  
James E. Smolen ◽  
Kenneth J. Balazovich ◽  
Rebecca R. Sandborg ◽  
Laurence A. Boxer
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Plasma Membrane Fraction ◽  
Calcium Dependent

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

Additive effect of contractions and insulin on GLUT-4 translocation into the sarcolemma

1994 ◽  
Vol 77 (4) ◽  
pp. 1597-1601 ◽  
Author(s):  
J. Gao ◽  
J. Ren ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Group ◽  
Intracellular Membrane ◽  
Glut 4 ◽  
Incremental Effect ◽  
Glut 4 Translocation

The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.

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