scholarly journals Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers.

1994 ◽  
Vol 125 (4) ◽  
pp. 733-741 ◽  
Author(s):  
L Saucan ◽  
G E Palade
Keyword(s):  
Golgi Complex ◽  
Magnetic Beads ◽  
Glucose Transporter ◽  
Sucrose Density Gradient ◽  
Secretory Proteins ◽  
Sds Page ◽  
Vesicular Carriers ◽  
Glandular Cells ◽  
Rat Livers

From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carriers operating between the Golgi complex and the basolateral (sinusoidal) plasmalemma. The starting preparation is a Golgi light fraction (GLF) isolated by flotation in a discontinuous sucrose density gradient and processed through immunoisolation on magnetic beads coated with an antibody against the last 11 aa. of the pIgA-R tail. GLF and the ensuing subfractions (bound vs nonbound) were lysed, and the lysates processed through immunoprecipitation with anti-pIgA-R and anti-albumin antibodies followed by radioactivity counting, SDS-PAGE, and fluorography. The recovery of newly synthesized pIgA-R was > 90% and the distribution was 90% vs 10% in the bound vs nonbound subfractions, respectively. Albumin radioactivity was recovered to approximately 80%, with 20% and 80% in bound vs nonbound subfractions, respectively. Other proteins studied were: (a) secretory-apolipoprotein-B, prothrombin, C3 component of the complement, and caeruloplasmin; (b) membrane-transferrin receptor, EGR-receptor, asialoglycoprotein receptor, and the glucose transporter. In all the experiments we have performed, the secretory proteins distributed up to 85% in the nonbound subfraction (large secretory vacuoles), whereas the membrane proteins were segregated up to 95% in the bound subfraction (small vesicular carriers). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmalemma by separate vesicular carriers as in glandular cells capable of constitutive and regulated secretion.

scholarly journals The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

2003 ◽  
Vol 162 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Pierre Morsomme ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Golgi Complex ◽  
Protein Sorting ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Cargo Protein ◽  
Conserved Oligomeric Golgi Complex ◽  
Sorting Process ◽  
Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

scholarly journals Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.

1983 ◽  
Vol 97 (5) ◽  
pp. 1582-1591 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Secretory Component ◽  
Bile Canaliculus ◽  
Sds Page ◽  
Iga Antibody ◽  
Co Precipitation ◽  
Kinetics Of

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1973 ◽  
Vol 58 (2) ◽  
pp. 436-462 ◽  
Author(s):  
Gert Kreibich ◽  
Pascale Debey ◽  
David D. Sabatini
Keyword(s):  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Transport Systems ◽  
Secretory Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Detergent Concentration ◽  
Doc Concentration ◽  
Microsomal Membranes

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocytes after bile duct ligation

1991 ◽  
Vol 98 (2) ◽  
pp. 205-216 ◽  
Author(s):  
J.M. Larkin ◽  
G.E. Palade
Keyword(s):  
Bile Duct ◽  
Serum Proteins ◽  
Bile Duct Ligation ◽  
Rat Hepatocytes ◽  
Frozen Sections ◽  
Transmission Electron ◽  
Duct Ligation ◽  
Sds Page ◽  
Vesicular Carriers

In rat hepatocytes, transcytotic vesicular carriers transport the mature 120 × 10(3) Mr form of the polymeric IgA receptor (pIgA-R), with or without its ligand, pIgA, from the sinusoidal to the biliary plasmalemma, where the ectodomain of the receptor is cleaved to produce an 80 × 10(3) Mr fragment that is secreted into the bile. Here we show that cholestasis induced by bile duct ligation results in the accumulation of transcytotic carriers, identified by the 120 × 10(3) Mr pIgA-R and pIgA, in the pericanalicular cytoplasm of hepatocytes. To determine the extent of pIgA-R accumulation, hepatic total microsomes (TM) were prepared from control and cholestatic rats. Solubilized TM proteins were separated by SDS-PAGE and receptor forms were detected by immunoblotting and autoradiography. Quantitative densitometry of these autoradiograms showed that after duct ligation the 120 × 10(3) Mr receptor accumulated to a level approximately threefold higher than the control. Concomitantly, immunologically related, novel 124, 90 and 80 × 10(3) Mr proteins (cholestatic antigens) became detectable. Immunoblot analyses of biliary and serum proteins showed that cholestasis resulted in: (1) a marked decrease in the concentrations of the 80 × 10(3) Mr receptor and pIgA in the bile, whereas albumin concentrations remained at control levels; and (2) a marked increase in the concentration of the 80 × 10(3) Mr receptor in the serum. Positive sites for pIgA-R were localized to the pericanalicular cytoplasm of hepatocytes by indirect immunofluorescence on semithin frozen sections in cholestatic hepatocytes. The sites were more numerous and the positive signal stronger than in controls. One day post-ligation, pIgA-positive sites were located to the same pericanalicular cytoplasm of hepatocytes; by three days, however, most pIgA appeared in sinusoidal endothelia and Kupffer cells. To validate the vesicular character of the receptor-positive sites, sham-operated and cholestatic livers were processed for either transmission electron microscopy (TEM) or immunogold localization of receptors on thin frozen sections. TEM verified the accumulation of pericanalicular vesicles in cholestatic hepatocytes. Immunogold tests localized pIgA-R to pleiomorphic, pericanalicular vesicles, which were increased in number, size and concentration of antigenic sites in cholestatic hepatocytes. These findings indicate that bile duct ligation provides a method for manipulating the in vivo transcytotic pathway and for accumulating previously unstudied transcytotic carriers in hepatocytes.

scholarly journals Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

1985 ◽  
Vol 100 (4) ◽  
pp. 1255-1261 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Companion Paper ◽  
Secretory Component ◽  
Cell Fractionation ◽  
Vesicular Carriers ◽  
Hepatic Proteins ◽  
Intact Rats ◽  
Er Membrane

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.

scholarly journals Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum.

1995 ◽  
Vol 128 (1) ◽  
pp. 29-38 ◽  
Author(s):  
P S Kim ◽  
P Arvan
Keyword(s):  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Disulfide Bonds ◽  
Secretory Proteins ◽  
Chaperone Function ◽  
Er Chaperone ◽  
Sds Page ◽  
Membrane Bound ◽  
Fraction Bound

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.

Ultrastructural Aspects of Golgi Complex Function in Parathyroid Cells of Winter Frogs

1973 ◽  
Vol 31 ◽  
pp. 680-681
Author(s):  
William J. Dougherty
Keyword(s):  
Golgi Complex ◽  
Secretory Granules ◽  
Complex Function ◽  
Secretory Activity ◽  
Secretory Proteins ◽  
Perivascular Spaces ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Ca Ions ◽  
Regulation Of Secretion

The regulation of secretion in exocrine and endocrine cells has long been of interest. Electron microscopic and other studies have demonstrated that secretory proteins synthesized on ribosomes are transported by the rough ER to the Golgi complex where they are concentrated into secretory granules. During active secretion, secretory granules fuse with the cell membrane, liberating and discharging their contents into the perivascular spaces. When secretory activity is suppressed in anterior pituitary cells, undischarged secretory granules may be degraded by lysosomes. In the parathyroid gland, evidence indicates that the level of blood Ca ions regulates both the production and release of parathormone. Thus, when serum Ca is low, synthesis and release of parathormone are both stimulated; when serum Ca is elevated, these processes are inhibited.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

GLUT2 and hexokinase control proximodistal gradient of intestinal glucose metabolism in the newborn pig

1997 ◽  
Vol 272 (6) ◽  
pp. G1530-G1539 ◽  
Author(s):  
C. Cherbuy ◽  
B. Darcy-Vrillon ◽  
L. Posho ◽  
P. Vaugelade ◽  
M. T. Morel ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Glucose Transporter ◽  
Glucose Utilization ◽  
Specific Effect ◽  
Glucose Consumption ◽  
Utilization Rate ◽  
Glucose Turnover ◽  
Proximal Jejunum ◽  
A Site

We have reported previously that a high glycolytic capacity develops soon after birth in enterocytes isolated from suckling newborn pigs. In the present work, we investigated whether such metabolic changes could affect intestinal glucose utilization in vivo and examined possible variations in glucose metabolism along the small intestine. Glucose utilization by individual tissues was assessed using the 2-deoxyglucose technique. The overall glucose utilization rate was doubled in suckling vs. fasting 2-day-old pigs because of significantly higher rates in all tissues studied, except for the brain. In parallel, enterocytes were isolated from the proximal, medium, or distal jejunoileum of newborn vs. 2-day-old pigs and assessed for their capacity to utilize, transport, and phosphorylate glucose. Intestinal glucose consumption accounted for approximately 15% of glucose turnover rate in suckling vs. 8% in fasting pigs. Moreover, there was a proximal-to-distal gradient of glucose utilization in the intestinal mucosa of suckling pigs. Such a gradient was also evidenced on isolated enterocytes. The stimulation of both hexokinase activity (HK2 isoform) and basolateral glucose transporter (GLUT2), as observed in the proximal jejunum, could account for such a site-specific effect of suckling.

scholarly journals Glucolipotoxicity and GLP-1 secretion

2021 ◽  
Vol 9 (1) ◽  
pp. e001905
Author(s):  
Jung-Hee Hong ◽  
Dae-Hee Kim ◽  
Moon-Kyu Lee
Keyword(s):  
Glucose Transporter ◽  
Cell Function ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Simultaneous Treatment ◽  
L Cells ◽  
Triglyceride Accumulation

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide (GLP-1) secretion, we generated glucolipotoxic conditions in human NCI-H716 enteroendocrine cells using either 5 or 25 mM glucose with or without 500 µM palmitate for 72 hours. For in vivo study, we have established a chronic nutrient infusion model in the rat. Serial blood samples were collected for 2 hours after the consumption of a mixed meal to evaluate insulin sensitivity and β-cell function.ResultsChronic glucolipotoxic conditions decreased GLP-1 secretion and the expressions of pCREB, pGSK3β, β-catenin, and TCF7L2 in NCI-H716 cells. Glucolipotoxicity conditions reduced glucose transporter expression, glucose uptake, and nicotinamide-adenine dinucleotide phosphate (NADPH) levels in L-cells, and increased triglyceride accumulation. In contrast, PPARα and ATP levels were reduced, which correlated well with decreased levels of SUR1 and Kir6.2, cAMP contents and expressions of pCAMK2, EPAC and PKA. We also observed an increase in reactive oxygen species production, UCP2 expression and Complex I activity. Simultaneous treatment with insulin restored the GLP-1 secretion. Glucolipotoxic conditions decreased insulin secretion in a time-dependent manner in INS-1 cells, which was recovered with exendin-4 cotreatment. Glucose and SMOFlipid infusion for 6 hours decreased GLP-1 secretion and proglucagon mRNA levels as well as impaired the glucose tolerance, insulin and C-peptide secretion in rats.ConclusionThese results provide evidence for the first time that glucolipotoxicity could affect GLP-1 secretion through changes in glucose and lipid metabolism, gene expressions, and proglucagon biosynthesis and suggest the interrelationship between glucolipotoxicities of L-cells and β-cells which develops earlier than that of L-cells.

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