scholarly journals Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2

1993 ◽  
Vol 294 (3) ◽  
pp. 735-743 ◽  
Author(s):  
S Benjannet ◽  
N Rondeau ◽  
L Paquet ◽  
A Boudreault ◽  
C Lazure ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Brefeldin A ◽  
Pulse Labelling ◽  
Prohormone Convertases ◽  
Post Translational Modifications ◽  
Intracellular Degradation ◽  
Golgi Compartment ◽  
Carbonyl Cyanide ◽  
Insulinoma Cells

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

1998 ◽  
Vol 337 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Adrie J. M. VERHOEVEN ◽  
Bernadette P. NEVE ◽  
Hans JANSEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Trafficking ◽  
De Novo ◽  
Hepatic Lipase ◽  
Brefeldin A ◽  
Apparent Increase ◽  
Triacylglycerol Hydrolase ◽  
Oligosaccharide Processing ◽  
Carbonyl Cyanide ◽  
Catalytically Active

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of α1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8±2.1-fold higher (mean±S.D., n = 7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3±0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose–heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7±0.3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not α1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

scholarly journals Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

2004 ◽  
Vol 15 (4) ◽  
pp. 1843-1852 ◽  
Author(s):  
Magnus A. B. Axelsson ◽  
Graham Warren
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Fluorescence Microscopy ◽  
Cell Line ◽  
Lateral Diffusion ◽  
Brefeldin A ◽  
Pharmacological Analysis ◽  
Golgi Vesicles ◽  
Video Fluorescence Microscopy ◽  
Golgi Compartment

Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes.

scholarly journals GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

1993 ◽  
Vol 123 (6) ◽  
pp. 1687-1694 ◽  
Author(s):  
B Thorens ◽  
N Gérard ◽  
N Dériaz
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Beta Cells ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Surface Expression ◽  
Trans Golgi Network ◽  
Intracellular Vesicles ◽  
Insulinoma Cells ◽  
Golgi Network

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

scholarly journals Proteomic Advances in Milk and Dairy Products

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3832
Author(s):  
Rubén Agregán ◽  
Noemí Echegaray ◽  
María López-Pedrouso ◽  
Radwan Kharabsheh ◽  
Daniel Franco ◽  
...  
Keyword(s):  
Dairy Products ◽  
Two Dimensional Gel Electrophoresis ◽  
Vast Number ◽  
Post Translational Modifications ◽  
Fundamental Parameters ◽  
Complex Fluid ◽  
Proteins And Peptides ◽  
Milk And Dairy Products

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.

scholarly journals Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chaitanya Erady ◽  
Adam Boxall ◽  
Shraddha Puntambekar ◽  
N. Suhas Jagannathan ◽  
Ruchi Chauhan ◽  
...  
Keyword(s):  
Transcript Level ◽  
Open Reading Frames ◽  
The Cancer Genome Atlas ◽  
Small Subset ◽  
Cancer Tissue ◽  
Post Translational Modifications ◽  
Cancer Genome Atlas ◽  
Systematic Identification ◽  
Reading Frames

AbstractUncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

Sign in / Sign up

Export Citation Format

Share Document