Abstract 390: VLDL Exits From The Endoplasmic Reticulum In A Specialized VLDL-transporting-vesicle (VTV) In Rat Primary Hepatocytes.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Shadab A Siddiqi
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Density Lipoprotein ◽  
Protein Composition ◽  
Secretory Protein ◽  
Proteinase K ◽  
Primary Hepatocytes ◽  
Density Region ◽  
Continuous Sucrose Gradient

Transport of very low-density lipoprotein (VLDL) from its site of synthesis, the endoplasmic reticulum (ER), to the Golgi is required for its eventual secretion from hepatocytes. This step represents a potential therapeutic target in controlling VLDL export and thus control of its metabolic derivative, LDL, the major carrier of cholesterol and determinant of atherosclerosis. The present study was designed to understand how VLDL exits from the ER at the molecular level. We developed an in vitro ER-budding assay in which rat liver ER (500 ug) was pre-loaded with 14 C-triacylglycerol (TAG) to mark VLDL and 3 H-proteins to mark newly synthesized proteins. The ER was incubated with rat liver cytosol (1 mg), GTP, and ATP at 37 o C for 30 min. The reaction mix was fractionated on a continuous sucrose gradient and the distribution of 14 C-TAG and 3 H-protein across the gradient was determined. 14 C-TAG was found in the light density region of the gradient, the expected place for TAG-rich VLDL carrying vesicles whereas 3 H-proteins appeared in the mid portion, the expected place in the gradient for protein vesicles. We examined the distribution of apolipoprotein B100 (apoB100), a marker for VLDL and albumin (a typical liver secretory protein) across the same gradient by Western blotting. As expected, apoB100 was distributed in light fractions whereas albumin was mainly in the mid portion of the gradient. These data show that VLDL and albumin are transported in vesicles of differing density. We hypothesize that a specialized vesicle is utilized for VLDL transport, which we name the VLDL-transporting-vesicle (VTV). Our results show that the release of VTV from rat liver ER requires cytosol, GTP, Sar1 (a GTPase), ATP, and incubation at 37 o C. VTV was sealed as judged by apoB100 signal post proteinase K treatment. VTVs concentrate ApoB100, Sar1, and exclude ER-resident protein calnexin. VTV fuses with liver cis -Golgi and delivers its cargo, VLDL, to the Golgi lumen. 2D-gels and electron microscopy data reveal that VTVs are different in their protein composition and are larger in size when compared to albumin carrying vesicles. In conclusion, we have identified and characterized a new ER-derived vesicle, VTV, which transports nascent VLDL from the ER to the Golgi in primary hepatocytes.

VLDL exits from the endoplasmic reticulum in a specialized vesicle, the VLDL transport vesicle, in rat primary hepatocytes

2008 ◽  
Vol 413 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Shadab A. Siddiqi
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Rat Hepatocytes ◽  
Proteinase K ◽  
Density Fractions ◽  
Transport Vesicles ◽  
Transport Vesicle

The movement of VLDL [very-LDL (low-density lipoprotein)] from the ER (endoplasmic reticulum) to the Golgi is required for its eventual secretion from hepatocytes and represents a potential target in controlling elevated concentrations of its metabolite LDL, the major determinant of atherosclerosis. To study this process, an in vitro ER-budding assay was developed to examine the generation of the VTV (VLDL transport vesicle) and PTV (protein transport vesicles) using ER isolated from [14C]TAG (triacylglycerol) and [3H]protein-labelled primary rat hepatocytes. VTVs do not contain albumin, as determined by immunoblots. VTVs were distributed in light-density fractions, whereas PTVs were mainly in the mid-portion of the sucrose gradient. Electron microscopy revealed that VTVs were larger (∼100–120 nm) in size than PTVs (∼55–70 nm). ER from 0.4 mM OA (oleic acid)-treated hepatocytes budded VTVs of a lighter density as compared with VTVs budded from ER of 0.1 mM or 0.004 mM OA-treated hepatocytes. The generation of VTVs from rat hepatic ER required cytosol, ATP, Sar1 (a GTPase) and incubation at 37 °C. Proteinase K treatment did not degrade the VTV cargo protein, apoB100 (apolipoprotein 100), indicating that VTVs were sealed. Immunoblots showed that VTV concentrated apoB100, Sar1 and rSec22b, and excluded albumin and calnexin. VTVs were shown to fuse with cis-Golgi and delivered their cargo to the Golgi lumen, as determined by in vitro fusion, and acquired endoglycosidase H resistance. These results suggest that a new ER-derived transport vesicle (VTV) has been identified and characterized which transports nascent VLDL from the hepatic ER to the Golgi.

scholarly journals Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

1982 ◽  
Vol 93 (1) ◽  
pp. 144-154 ◽  
Author(s):  
L Marzella ◽  
J Ahlberg ◽  
H Glaumann
Keyword(s):  
Proteolytic Activity ◽  
Rat Liver ◽  
Biochemical Characterization ◽  
Secretory Granules ◽  
Density Lipoprotein ◽  
Marker Enzymes ◽  
Cell Organelles ◽  
Protein Ratio ◽  
Autophagic Vacuoles

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Uptake of cholesterol from the very low density lipoprotein or its remnants by the perfused rat liver

1981 ◽  
Vol 59 (6) ◽  
pp. 447-453 ◽  
Author(s):  
Simon-Pierre Noël ◽  
David Rubinstein
Keyword(s):  
Rat Liver ◽  
Lipid Composition ◽  
Low Density Lipoprotein ◽  
Liver Perfusion ◽  
Density Lipoprotein ◽  
Hepatic Uptake ◽  
Low Density ◽  
Perfused Liver ◽  
Very Low Density Lipoproteins

[3H]Cholesterol labelled very low density lipoproteins ([3H]chol-VLDL) were prepared to study the hepatic uptake of cholesterol associated with VLDL and its remnants in the perfused liver system. [3H]Chol-VLDL was incubated with rat postheparin plasma to produce labelled remnants in vitro. The degree of lipolysis of [3H]chol-VLDL depended on the ratio of triacylglycerols to lipase in the incubation medium. Therefore, the produced remnant of d < 1.019 g∙mL−1 had a variable lipid composition depending on the degree of lipolysis. [3H]Chol-VLDL or its remnants were added to liver perfusate and the radioactivity remaining in the perfusate was measured. The kinetic disappearance of [3H]chol-VLDL and its remnants in the perfused liver system indicated that remnant of d < 1.019 g∙mL−1 was taken up by the liver faster than the original VLDL preparation (t1/2 of 8 min vs. 51 min). Appearance of the label in bile during the perfusion was significantly faster when livers were perfused with [3H]chol-VLDL remnants as opposed to uncatabolized [3H]chol-VLDL.The results indicate that first of all, VLDL remnants produced in vitro and reisolated at density less than 1.019 g∙mL−1 do not have a fixed lipid composition but a rather variable one depending on the degree of lipolysis. Secondly, the rat liver may preferentially recognize this VLDL remnant of d < 1.019 g∙mL−1 and take it up more readily than uncatabolized VLDL. Finally when equimolar amount of cholesterol from VLDL or VLDL remnants are circulated in the liver perfusion, the VLDL remnants convey a significantly greater mass of cholesterol to the bile.

scholarly journals A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

2005 ◽  
Vol 79 (8) ◽  
pp. 5163-5173 ◽  
Author(s):  
Els Wessels ◽  
Daniël Duijsings ◽  
Richard A. Notebaart ◽  
Willem J. G. Melchers ◽  
Frank J. M. van Kuppeveld
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Model ◽  
Ectopic Expression ◽  
Rna Replication ◽  
Secretory Protein ◽  
Viral Rna ◽  
Rich Region ◽  
Golgi Transport ◽  
Proline Rich Region

ABSTRACT The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

scholarly journals Altered hepatic catabolism of low-density lipoprotein subjected to lipid peroxidation in vitro

1994 ◽  
Vol 297 (3) ◽  
pp. 573-579 ◽  
Author(s):  
W L Stone ◽  
M Heimberg ◽  
R L Scott ◽  
I LeClair ◽  
H G Wilcox
Keyword(s):  
Lipid Peroxidation ◽  
Rat Liver ◽  
Low Density Lipoprotein ◽  
Liver Perfusion ◽  
Density Lipoprotein ◽  
Low Density ◽  
Perfusion System ◽  
Oxidatively Modified ◽  
Generating System

Recent evidence suggests that oxidatively modified forms of low-density lipoprotein (LDL) may be particularly atherogenic. In this investigation, the catabolism of human LDL modified by lipid peroxidation in vitro was studied with a recirculating rat liver perfusion system. A dual-labelling technique was used that permitted native LDL and modified LDL to be studied simultaneously in the liver perfusion system. Native human LDL was found to have a fractional catabolic rate (FCR) of 1.00 +/- 0.21%/h, in agreement with other investigators. Subjecting LDL to oxidation for 12 h in the presence of 30 microM FeEDTA did not significantly affect its FCR. LDL treated with a superoxide-generating system (xanthine oxidase, hypoxanthine, O2) in the presence of 30 microM FeEDTA did, however, show a significant increase in FCR (3.23 +/- 0.19%/h). The hepatic uptakes of native LDL and LDL oxidized with FeEDTA+O2 were similar, but both were significantly lower than the hepatic uptake of LDL treated with the superoxide-radical-generating system. The proteolysis of LDL with pancreatin did not influence either its susceptibility to oxidation or its FCR. LDL oxidation resulted in the preferential loss of alpha-tocopherol rather than gamma-tocopherol. These data indicate that the rat liver effectively catabolizes LDL oxidatively modified by treatment with the superoxide-generating system. Furthermore, our results suggest that only very low plasma levels of highly oxidized LDL could be found under conditions in vivo. The liver may therefore play a major role in protecting the arterial vasculature from highly atherogenic forms of LDL.

scholarly journals Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1974 ◽  
Vol 142 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Susan C. Davison ◽  
Eric D. Wills
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Turnover Rate ◽  
Total Phospholipid ◽  
Transient Increase ◽  
Phospholipid Synthesis ◽  
Phosphatidylethanolamine Methyltransferase ◽  
Phosphatidylcholine Synthesis

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [32P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [32P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine–phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [32P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [14C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [32P]orthophosphate and [14C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [32P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [32P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.

scholarly journals Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

Sign in / Sign up

Export Citation Format

Share Document