scholarly journals Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane

1996 ◽  
Vol 315 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Xavier BOSSUYT ◽  
Norbert BLANCKAERT
Keyword(s):  
Endoplasmic Reticulum ◽  
Glucuronic Acid ◽  
Endoplasmic Reticulum Membrane ◽  
Acid Uptake ◽  
Physiological Importance ◽  
Cytosolic Side ◽  
Udp Glucuronosyltransferase ◽  
Exogenous Compounds ◽  
Stimulation Of ◽  
Er Membrane

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xylose produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exerted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

scholarly journals Mechanism of stimulation of microsomal UDP-glucuronosyltransferase by UDP-N-acetylglucosamine

1995 ◽  
Vol 305 (1) ◽  
pp. 321-328 ◽  
Author(s):  
X Bossuyt ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Glucuronic Acid ◽  
Carrier System ◽  
Transport Pathways ◽  
Weak Inhibitor ◽  
Cytosolic Side ◽  
In Trans ◽  
Udp Glucuronosyltransferase ◽  
Carrier Systems ◽  
Stimulation Of

We propose the existence in rat liver endoplasmic reticulum (ER) of two asymmetric carrier systems. One system couples UDP-N-acetylglucosamine (UDPGlcNAc) transport to UDP-glucuronic acid (UDPGlcA) transport. When UDPGlcNAc was presented at the cytosolic side of the ER, it then acted as a weak inhibitor of UDPGlcA uptake. By contrast, UDPGlcNAc produced a forceful trans-stimulation of microsomal UDPGlcA uptake when it was present within the lumen of the ER. Likewise, cytosolic UDPGlcA strongly trans-stimulated efflux of intravesicular UDPGlcNAc, whereas cytosolic UDPGlcNAc was ineffective in trans-stimulating efflux of UDPGlcA. A second asymmetric carrier system couples UDPGlcNAc transport to UMP transport. Microsomal UDPGlcNAc influx was markedly stimulated by UMP present inside the microsomes. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP, indicating that UMP exerted its effect on UDPGlcNAc uptake by trans-stimulation from the lumenal side of the ER membrane. Contrariwise, extravesicular UMP only minimally trans-stimulated efflux of intramicrosomal UDPGlcNAc. It is widely accepted that UDPGlcNAc acts as a physiological activator of hepatic glucuronidation, but the mechanism of this effect has remained elusive. Based on our findings, we propose a model in which the interaction of two asymmetric transport pathways, i.e. UDPGlcA influx coupled to UDPGlcNAc efflux and UDPGlcNAc influx coupled to UMP efflux, combined with intravesicular metabolism of UDPGlcA, forms a mechanism that leads to stimulation of glucuronidation by UDPGlcNAc.

scholarly journals Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles

1996 ◽  
Vol 315 (1) ◽  
pp. 171-176 ◽  
Author(s):  
Gábor BÁNHEGYI ◽  
László BRAUN ◽  
Paola MARCOLONGO ◽  
Miklós CSALA ◽  
Rosella FULCERI ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Glucuronic Acid ◽  
Udp Glucuronosyltransferase ◽  
Luminal Compartment ◽  
Loading Procedure ◽  
Stimulation Of

The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze–thawing method. It was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP-glucuronic acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetylglucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports UDP-glucuronic acid into the lumen of the endoplasmic reticulum.

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 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 Mammalian Lass6 and its related family members regulate synthesis of specific ceramides

2005 ◽  
Vol 390 (1) ◽  
pp. 263-271 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Endoplasmic Reticulum ◽  
Cultured Cells ◽  
Family Members ◽  
Terminal Region ◽  
Proteinase K ◽  
Endoplasmic Reticulum Membrane ◽  
Substrate Preferences ◽  
Luminal Side ◽  
Ceramide Synthesis ◽  
Cytosolic Side

The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.

scholarly journals Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

1996 ◽  
Vol 7 (5) ◽  
pp. 769-789 ◽  
Author(s):  
A J Koning ◽  
C J Roberts ◽  
R L Wright
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Endogenous Expression ◽  
Coa Reductase ◽  
Er Membrane

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

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