Endoplasmin is a reticuloplasmin

1988 ◽  
Vol 90 (3) ◽  
pp. 485-491
Author(s):  
G.L. Koch ◽  
D.R. Macer ◽  
F.B. Wooding
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Immunogold Labelling ◽  
Soluble Form ◽  
Intact Molecule ◽  
Carboxy Terminal ◽  
Triton X ◽  
Separation Test

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

scholarly journals Infectious Bronchitis Virus 3a Protein Localizes to a Novel Domain of the Smooth Endoplasmic Reticulum

2005 ◽  
Vol 79 (10) ◽  
pp. 6142-6151 ◽  
Author(s):  
Amanda R. Pendleton ◽  
Carolyn E. Machamer
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Infectious Bronchitis Virus ◽  
Smooth Endoplasmic Reticulum ◽  
Integral Membrane Protein ◽  
Open Reading Frames ◽  
Infectious Bronchitis ◽  
Infected Cells ◽  
Group 3 ◽  
Small Open Reading Frames

ABSTRACT All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.

scholarly journals Purification and properties of a neurotensin-degrading endopeptidase from pig brain

1991 ◽  
Vol 276 (3) ◽  
pp. 583-591 ◽  
Author(s):  
P E Millican ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Membrane Protein ◽  
Hydrophobic Interactions ◽  
Total Activity ◽  
Integral Membrane Protein ◽  
Major Band ◽  
Pig Brain ◽  
Purification And Properties ◽  
Endopeptidase Activity ◽  
Triton X ◽  
Endopeptidase 24.15

Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and

scholarly journals UDP-Galactose:Ceramide Galactosyltransferase Is a Class I Integral Membrane Protein of the Endoplasmic Reticulum

1998 ◽  
Vol 273 (40) ◽  
pp. 25880-25888 ◽  
Author(s):  
Hein Sprong ◽  
Boudewijn Kruithof ◽  
Richtje Leijendekker ◽  
Jan Willem Slot ◽  
Gerrit van Meer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Class I

scholarly journals Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

1985 ◽  
Vol 33 (8) ◽  
pp. 813-820 ◽  
Author(s):  
K L Goldenthal ◽  
K Hedman ◽  
J W Chen ◽  
J T August ◽  
M C Willingham
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Membrane Glycoproteins ◽  
Animal Cells ◽  
Cytoplasmic Proteins ◽  
Membrane Antigens ◽  
Endocytic Vesicles ◽  
Triton X ◽  
Nonionic Detergents

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

scholarly journals The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N

1985 ◽  
Vol 231 (2) ◽  
pp. 445-449 ◽  
Author(s):  
R Matsas ◽  
S L Stephenson ◽  
J Hryszko ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Phase Separation ◽  
Membrane Protein ◽  
Total Activity ◽  
Aminopeptidase N ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Rich Phase ◽  
Triton X

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 × 10(−7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 × 10(−6) M) than for the other two forms (IC50 = 1.6 × 10(−5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 × 10(−5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 × 10(−4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.

scholarly journals Erv26p Directs Pro-Alkaline Phosphatase into Endoplasmic Reticulum–derived Coat Protein Complex II Transport Vesicles

2006 ◽  
Vol 17 (11) ◽  
pp. 4780-4789 ◽  
Author(s):  
Catherine A. Bue ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Protein ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Copii Vesicles

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Δ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.

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