scholarly journals 5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

1993 ◽  
Vol 178 (6) ◽  
pp. 1935-1946 ◽  
Author(s):  
J W Woods ◽  
J F Evans ◽  
D Ethier ◽  
S Scott ◽  
P J Vickers ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Resting Cells ◽  
Human Leukocytes ◽  
Ultrathin Frozen Sections

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

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.

scholarly journals CHARACTERIZATION OF RAT LIVER SUBCELLULAR MEMBRANES

1974 ◽  
Vol 60 (2) ◽  
pp. 460-472 ◽  
Author(s):  
David H. DeHeer ◽  
Merle S. Olson ◽  
R. Neal Pinckard
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Membrane Fraction ◽  
Cross Reactivity ◽  
Selective Absorption ◽  
Mitochondrial Membranes ◽  
Subcellular Membrane ◽  
Hepatocellular Necrosis

The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes.

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 Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

1996 ◽  
Vol 315 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Callum LIVINGSTONE ◽  
David E. JAMES ◽  
Jacqueline E. RICE ◽  
David HANPETER ◽  
Gwyn W. GOULD
Keyword(s):  
Plasma Membrane ◽  
Okadaic Acid ◽  
Transferrin Receptor ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Type I ◽  
Intracellular Membrane ◽  
Intracellular Compartment ◽  
Endosomal System

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the co-localization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of co-localization of these proteins. Using a technique to cross-link and render insoluble (‘ablate’) intracellular compartments containing the TfR by means of a transferrin–horseradish peroxidase conjugate (Tf–HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf–HRP for 3 h at 37 °C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf–HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative, TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

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.

scholarly journals Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

2020 ◽  
Author(s):  
David J. Thaller ◽  
Danqing Tong ◽  
Christopher J. Marklew ◽  
Sapan Borah ◽  
Barbara Ciani ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Specific Binding ◽  
Binding Activity ◽  
Nuclear Pore ◽  
Membrane Remodeling ◽  
Functional Importance ◽  
Domain Protein

AbstractMechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding activity in the nuclear envelope-specific ESCRT, Chm7, in budding yeast. PA-binding is mediated through a conserved hydrophobic stretch of amino acids, which confers specific binding to the inner nuclear membrane (INM). This INM-binding is independent but nonetheless required for interaction with the LAP2-emerin-MAN1 (LEM) domain protein, Heh1 (LEM2). Consistent with the functional importance of PA-binding, mutation of this region inhibits recruitment of Chm7 to the INM and abolishes Chm7 function in the context of nuclear envelope herniations or “blebs” that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that PA accumulates at nuclear envelope herniations. We suggest that local control of PA metabolism is important for ensuring productive nuclear envelope remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

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 Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells

1994 ◽  
Vol 303 (2) ◽  
pp. 481-487 ◽  
Author(s):  
V Le Cabec ◽  
I Maridonneau-Parini
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Membrane Fraction ◽  
Immunofluorescence Microscopy ◽  
Resting Cells ◽  
Dependent Manner ◽  
Intact Cells ◽  
Cytoplasmic Granules ◽  
Calcium Dependent ◽  
Specific Granules

Annexins are soluble proteins capable of binding to phospholipid membranes in a calcium-dependent manner. Annexin 3, a 33 kDa protein mainly expressed in neutrophils, aggregates granules in cell-free assays, and a 36 kDa variant of this protein, specifically expressed in monocytes, has recently been identified. To obtain further information on these proteins, we defined their subcellular localization in resting and activated cells by immunofluorescence microscopy. Both proteins were associated with cytoplasmic granules in resting cells. We obtained evidence to indicate that, in neutrophils which possess a heterogenous granule population, annexin 3 was more likely to be associated with the specific granules. In cells activated with phorbol 12-myristate 13-acetate or opsonized zymosan, the 33 kDa and 36 kDa proteins translocated to the plasma or the phagosome membrane. Upon stimulation with A23187, annexin 3 translocated to the plasma membrane only in neutrophils. We also report that while annexin 3 was associated with restricted membranes in intact cells, it binds indiscriminately to every membrane fraction in cell-free assay. In conclusion, association of both forms of annexin 3 with granules suggests that these proteins could be implicated in processes of granule fusion.

THE ORIGIN OF THE PECTIC LAYER OF THE CELL WALL OF SCENEDESMUS QUADRICAUDA

1965 ◽  
Vol 43 (12) ◽  
pp. 1549-1552 ◽  
Author(s):  
T. Bisalputra
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Basic Structure ◽  
Scenedesmus Quadricauda ◽  
Outer Nuclear Membrane ◽  
Dense Vesicles

The origin of the pectic layer of the cell wall of Scenedesmus quadricauda can be related to the activity of the nuclear envelope. During the four-nucleate stage, dense vesicles are formed by budding off from the outer nuclear membrane. These vesicles then migrate toward the plasma membrane and their contents are released from the cytoplasm by the process of reverse pinocytosis. From the released matrix the props and the net are organized; they become the easily recognized basic structure of the pectic layer of the cell wall.

Sign in / Sign up

Export Citation Format

Share Document