scholarly journals An investigation of the transverse topology of bilirubin UDP-glucuronosyltransferase in rat hepatic endoplasmic reticulum

1989 ◽  
Vol 259 (2) ◽  
pp. 617-620 ◽  
Author(s):  
S R Shepherd ◽  
S J Baird ◽  
T Hallinan ◽  
B Burchell
Keyword(s):  
Endoplasmic Reticulum ◽  
Sodium Deoxycholate ◽  
Immunoblot Analysis ◽  
Proteinase K ◽  
Transmembrane Topology ◽  
Working Model ◽  
Hepatic Microsomes ◽  
Udp Glucuronosyltransferase ◽  
Cytoplasmic Side

Bilirubin UDP-glucuronosyltransferase (UDPGT) activity in sealed hepatic microsomes from clofibrate-treated rats was highly latent and was fully expressed by disruption of vesicles with detergents. Antibodies raised against purified bilirubin UDPGT were used to study the transmembrane orientation of the protein to provide a molecular understanding of the UDPGT latency. Immunoblot analysis of sealed microsomes, and microsomes after treatment with proteinases, showed that only a small portion of the protein resides on the cytoplasmic side of the microsomal vesicles. Treatment of microsomes with sodium deoxycholate allowed subtilisin and proteinase K to cleave the transferase, causing loss of activity and the release of smaller immunodetectable peptides. Treatment of the purified bilirubin UDPGT with peptide N-glycosidase F indicated that the enzyme was a glycoprotein. A working model of the transmembrane topology of bilirubin UDPGT is described.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

scholarly journals Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2

2020 ◽  
Vol 318 (1) ◽  
pp. F135-F147
Author(s):  
Poorichaya Somparn ◽  
Chatikorn Boonkrai ◽  
Komgrid Charngkaew ◽  
Nusara Chomanee ◽  
Kenneth G. Hodge ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Ureteral Obstruction ◽  
Collecting Duct ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Sham Operation ◽  
Autophagic Degradation ◽  
Cell Cell

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

scholarly journals Investigation of the Endoplasmic Reticulum Localization of UDP-Glucuronosyltransferase 2B7 with Systematic Deletion Mutants

2019 ◽  
Vol 95 (5) ◽  
pp. 551-562 ◽  
Author(s):  
Yuu Miyauchi ◽  
Sora Kimura ◽  
Akane Kimura ◽  
Ken Kurohara ◽  
Yuko Hirota ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Deletion Mutants ◽  
Udp Glucuronosyltransferase

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 Borrelia burgdorferi Binds to, Invades, and Colonizes Native Type I Collagen Lattices

2004 ◽  
Vol 72 (6) ◽  
pp. 3138-3146 ◽  
Author(s):  
Maria C. Zambrano ◽  
Anastasia A. Beklemisheva ◽  
Anton V. Bryksin ◽  
Stuart A. Newman ◽  
Felipe C. Cabello
Keyword(s):  
Borrelia Burgdorferi ◽  
Type I Collagen ◽  
High Efficiency ◽  
Immunoblot Analysis ◽  
Proteinase K ◽  
Type I ◽  
Collagen Matrices ◽  
Proteinase K Treatment ◽  
Collagen Lattices

ABSTRACT Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between B. burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices. B. burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices, B. burgdorferi grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B. burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices. B. burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of B. burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B. burgdorferi to interact with intact collagen matrices. The high efficiency of binding of B. burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.

Sign in / Sign up

Export Citation Format

Share Document