Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo

1979 ◽  
Vol 57 (6) ◽  
pp. 625-638 ◽  
Author(s):  
Monique Behar-Bannelier ◽  
Rajendra N. Sharma ◽  
Robert K. Murray
Keyword(s):  
Endoplasmic Reticulum ◽  
Mouse Liver ◽  
Serum Proteins ◽  
Sodium Dodecyl ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Flow ◽  
Adult Mice ◽  
Coomassie Blue ◽  
Er Membrane

The use of L-[35S]methionine (500–700 Ci/mmol (1 Ci = 37 GBq)) for labelling the polypeptides of liver rough (R) and smooth (S) endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 μCi of the isotope and killed at various times (2 min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 μg protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.

scholarly journals Carrier-mediated transport of uridine diphosphoglucuronic acid across the endoplasmic reticulum membrane is a prerequisite for UDP-glucuronosyltransferase activity in rat liver

1997 ◽  
Vol 323 (3) ◽  
pp. 645-648 ◽  
Author(s):  
Xavier BOSSUYT ◽  
Norbert BLANCKAERT
Keyword(s):  
Endoplasmic Reticulum ◽  
Permeability Barrier ◽  
Endoplasmic Reticulum Membrane ◽  
Carrier Mediated Transport ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Stimulation Of ◽  
Er Membrane

UDP-glucuronosyltransferases (EC 2.4.1.17) is an isoenzyme family located primarily in the hepatic endoplasmic reticulum (ER) that displays latency of activity both in vitro and in vivo, as assessed respectively in microsomes and in isolated liver. The postulated luminal location of the active site of UDP-glucuronosyltransferases (UGTs) creates a permeability barrier to aglycone and UDP-GlcA access to the enzyme and implies a requirement for the transport of substrates across the ER membrane. The present study shows that the recently demonstrated carrier-mediated transport of UDP-GlcA across the ER membrane is required and rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes. We found that in both microsomes and permeabilized hepatocytes a gradual inhibition by N-ethylmaleimide (NEM) of UDP-GlcA transport into the ER produced a correspondingly increasing inhibition of 4-methylumbelliferone glucuronidation. That NEM selectively inhibited the UDP-GlcA transporter, without affecting intrinsic UGT activity, was demonstrated by showing that NEM had no effect on glucuronidation in microsomes or hepatocytes with permeabilized ER membrane. Additional evidence that UDP-GlcA transport is rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes was obtained by showing that gradual selective trans-stimulation of UDP-GlcA transport by UDP-GlcNAc, UDP-Xyl or UDP-Glc in each case produced correspondingly enhanced glucuronidation. Such stimulation of transport and glucuronidation was inhibited completely by NEM, which selectively inhibited UDP-GlcA transport.

scholarly journals Ceramide chain length–dependent protein sorting into selective endoplasmic reticulum exit sites

2020 ◽  
Vol 6 (50) ◽  
pp. eaba8237
Author(s):  
Sofia Rodriguez-Gallardo ◽  
Kazuo Kurokawa ◽  
Susana Sabido-Bozo ◽  
Alejandro Cortes-Gomez ◽  
Atsuko Ikeda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Chain Length ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Endoplasmic Reticulum Membrane ◽  
Lipid Chain ◽  
Secretory Transport ◽  
Dependent Protein ◽  
Cellular Compartmentalization

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length–based protein cargo sorting into selective export sites of the secretory pathway.

scholarly journals Spliced XBP1 Rescues Renal Interstitial Inflammation Due to Loss of Sec63 in Collecting Ducts

2019 ◽  
Vol 30 (3) ◽  
pp. 443-459 ◽  
Author(s):  
Yasunobu Ishikawa ◽  
Sorin Fedeles ◽  
Arnaud Marlier ◽  
Chao Zhang ◽  
Anna-Rachel Gallagher ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Genetic Model ◽  
Collecting Duct ◽  
Kidney Injury ◽  
Endoplasmic Reticulum Membrane ◽  
Double Knockout ◽  
Embryonic Mouse ◽  
Interstitial Inflammation ◽  
Collecting Ducts

BackgroundSEC63 encodes a resident protein in the endoplasmic reticulum membrane that, when mutated, causes human autosomal dominant polycystic liver disease. Selective inactivation of Sec63 in all distal nephron segments in embryonic mouse kidney results in polycystin-1–mediated polycystic kidney disease (PKD). It also activates the Ire1α-Xbp1 branch of the unfolded protein response, producing Xbp1s, the active transcription factor promoting expression of specific genes to alleviate endoplasmic reticulum stress. Simultaneous inactivation of Xbp1 and Sec63 worsens PKD in this model.MethodsWe explored the renal effects of postnatal inactivation of Sec63 alone or with concomitant inactivation of Xbp1 or Ire1α, specifically in the collecting ducts of neonatal mice.ResultsThe later onset of inactivation of Sec63 restricted to the collecting duct does not result in overt activation of the Ire1α-Xbp1 pathway or cause polycystin-1–dependent PKD. Inactivating Sec63 along with either Xbp1 or Ire1α in this model causes interstitial inflammation and associated fibrosis with decline in kidney function over several months. Re-expression of XBP1s in vivo completely rescues the chronic kidney injury observed after inactivation of Sec63 with either Xbp1 or Ire1α.ConclusionsIn the absence of Sec63, basal levels of Xbp1s activity in collecting ducts is both necessary and sufficient to maintain proteostasis (protein homeostasis) and protect against inflammation, myofibroblast activation, and kidney functional decline. The Sec63-Xbp1 double knockout mouse offers a novel genetic model of chronic tubulointerstitial kidney injury, using collecting duct proteostasis defects as a platform for discovery of signals that may underlie CKD of disparate etiologies.

scholarly journals Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

1984 ◽  
Vol 99 (3) ◽  
pp. 1076-1082 ◽  
Author(s):  
M G Rosenfeld ◽  
E E Marcantonio ◽  
J Hakimi ◽  
V M Ort ◽  
P H Atkinson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Posttranslational Modifications ◽  
Rat Hepatocytes ◽  
Direct Analysis ◽  
In Vitro Translation ◽  
Endoplasmic Reticulum Membrane ◽  
Pituitary Cells ◽  
Amino Terminal

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

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

1974 ◽  
Vol 61 (3) ◽  
pp. 789-807 ◽  
Author(s):  
Gert Kreibich ◽  
David D. Sabatini
Keyword(s):  
Serum Proteins ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Rat Serum ◽  
Coomassie Blue ◽  
Specific Subset ◽  
Low Levels ◽  
Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

scholarly journals Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects

2017 ◽  
Vol 214 (12) ◽  
pp. 3707-3729 ◽  
Author(s):  
Maria A. Rujano ◽  
Magda Cannata Serio ◽  
Ganna Panasyuk ◽  
Romain Péanne ◽  
Janine Reunert ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mouse Liver ◽  
Serum Proteins ◽  
Fat Body ◽  
Mammalian Target Of Rapamycin ◽  
Missense Mutations ◽  
Atpase Subunit ◽  
Altered Lipid Metabolism ◽  
Altered Lipid ◽  
Lysosomal Acidification

The biogenesis of the multi-subunit vacuolar-type H+-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN

1972 ◽  
Vol 52 (2) ◽  
pp. 231-245 ◽  
Author(s):  
Colvin M. Redman ◽  
M. George Cherian
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Rough Endoplasmic Reticulum ◽  
Serum Proteins ◽  
Rat Serum ◽  
Secretory Pathways ◽  
Specific Antisera ◽  
Microsome Fraction

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the 14C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.

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