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 Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

2001 ◽  
Vol 75 (22) ◽  
pp. 10651-10662 ◽  
Author(s):  
Lin Qu ◽  
Laura K. McMullan ◽  
Charles M. Rice
Keyword(s):  
Cultured Cells ◽  
Nonstructural Protein ◽  
Low Frequency ◽  
Endoplasmic Reticulum Membrane ◽  
Diarrhea Virus ◽  
Isolation And Characterization ◽  
Cytosolic Side ◽  
Infected Cells ◽  
Dominant Selectable Marker ◽  
Viral Diarrhea Virus

ABSTRACT Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

scholarly journals Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 848 ◽  
Author(s):  
Pingan Chang ◽  
Ling He ◽  
Yu Wang ◽  
Christoph Heier ◽  
Yijun Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Terminal Region ◽  
Neuropathy Target Esterase ◽  
Loss Of Function ◽  
Dynamic Interactions ◽  
Functional Regions ◽  
R Region

Neuropathy target esterase (NTE) is an endoplasmic reticulum (ER)-localized phospholipase that deacylates phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Loss-of-function mutations in the human NTE gene have been associated with a spectrum of neurodegenerative disorders such as hereditary spastic paraplegia, ataxia and chorioretinal dystrophy. Despite this, little is known about structure–function relationships between NTE protein domains, enzymatic activity and the interaction with cellular organelles. In the current study we show that the C-terminal region of NTE forms a catalytically active domain that exhibits high affinity for lipid droplets (LDs), cellular storage organelles for triacylglycerol (TAG), which have been recently implicated in the progression of neurodegenerative diseases. Ectopic expression of the C domain in cultured cells decreases cellular PC, elevates TAG and induces LD clustering. LD interactions of NTE are inhibited by default by a non-enzymatic regulatory (R) region with three putative nucleotide monophosphate binding sites. Together with a N-terminal TMD the R region promotes proper distribution of the catalytic C-terminal region to the ER network. Taken together, our data indicate that NTE may exhibit dynamic interactions with the ER and LDs depending on the interplay of its functional regions. Mutations that disrupt this interplay may contribute to NTE-associated disorders by affecting NTE positioning.

scholarly journals Syntaxin 17 Recruits ACSL3 to Lipid Microdomains in Lipid Droplet Biogenesis

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641983871
Author(s):  
Hana Kimura ◽  
Kohei Arasaki ◽  
Moe Iitsuka ◽  
Mitsuo Tagaya
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplet ◽  
Cholesterol Depletion ◽  
Endoplasmic Reticulum Membrane ◽  
Lipid Microdomains ◽  
Ceramide Synthesis ◽  
Detergent Resistant Membranes ◽  
A Minor ◽  
Ganglioside Gd3 ◽  
Neutral Lipid Biosynthesis

During lipid droplet (LD) formation, several key enzymes for neutral lipid biosynthesis, such as acyl-CoA synthetase 3 (ACSL3), translocate from the bilayer of the endoplasmic reticulum membrane or mitochondria-associated membrane to the monolayer surface of LDs. It has been recently shown that syntaxin 17 (Stx17) in cooperation with synaptosomal-associated protein of 23 kDa (SNAP23) facilitates the translocation of ACSL3 from the endoplasmic reticulum/mitochondria-associated membrane to LDs. In this study, we investigated whether lipid microdomains enriched in cholesterol and sphingolipids are important for the formation of LDs and the interaction of Stx17 with ACSL3 and SNAP23. Cholesterol depletion and blockage of ceramide synthesis by chemicals inhibited oleic acid (OA)-induced LD biogenesis and decreased the interaction of Stx17 with ACSL3 and SNAP23, whereas blockage of ganglioside GD3 synthesis by sialyltransferase knockdown interfered with LD biogenesis by affecting the interaction of Stx17 with SNAP23 but not ACSL3. Consistent with the requirement of GD3 in LD biogenesis, Stx17 was found to associate with GD3-containing membranes upon OA loading. SNAP23 and a minor fraction of Stx17 were found to reside in detergent-resistant membranes (DRMs), whereas OA treatment caused redistribution of ACSL3 and Stx17 to DRMs. Importantly, the redistribution of ACSL3 to DRMs was abrogated upon depletion of Stx17 or SNAP23. Taken together, our results highlight the importance of lipid microdomains enriched in cholesterol and sphingolipids as a platform for the interaction of Stx17 with ACSL3 and SNAP23 in LD biogenesis.

scholarly journals The C-terminal region of the hepatitis C virus E1 glycoprotein confers localization within the endoplasmic reticulum

1999 ◽  
Vol 80 (8) ◽  
pp. 1943-1947 ◽  
Author(s):  
Mike Flint ◽  
Jane A. McKeating
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Terminal Region ◽  
Golgi Compartment ◽  
Chimeric Molecules ◽  
Influenza Virus Haemagglutinin ◽  
Surface Fusion ◽  
Virus Haemagglutinin

Expression of the hepatitis C virus glycoprotein E1 in cultured cells localizes it to the endoplasmic reticulum, suggesting that E1 contains a signal mediating retention. Fusion of the C-terminal region of E1 to the ectodomain of CD4 prevented it from being transported to the cell surface. Fusion of this region of E1 resulted in localization of CD4 and influenza virus haemagglutinin chimeric molecules to a pre-medial Golgi compartment. This signal was present within E1 residues 311–383. Retention was not due to misfolding since the chimeric molecules did not form disulphide-linked aggregates indicative of misfolded proteins, and could be recognized by MAbs specific for conformational epitopes.

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.

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