Internalization and recycling to serotonin-containing granules of the 80K integral membrane protein exposed on the surface of secreting rat basophilic leukaemia cells

1989 ◽  
Vol 92 (4) ◽  
pp. 701-712
Author(s):  
J.S. Bonifacino ◽  
L. Yuan ◽  
I.V. Sandoval
Keyword(s):  
Membrane Protein ◽  
Secretory Granules ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Multivesicular Bodies ◽  
Pituitary Cells ◽  
Coated Pits ◽  
Golgi System ◽  
Rbl Cells ◽  
Natural Killer Lymphocytes

The 80K (80 × 10(3) Mr) integral membrane protein, first described in the secretory granules of rat basophilic leukaemia (RBL) cells, is also localized to lysosomes in these cells. The protein displays the same distribution in natural killer lymphocytes (RNK-7), wherein it codistributes with cytolysin in secretory granules. In contrast, the protein is absent from the endocrine and exocrine secretory granules of rat pancreatic acinar and pituitary cells, respectively, where it is confined to lysosomes. The protein colocalizes with lysosomal integral membrane proteins in all the cells studied, indicating that is largely restricted to secretory granules with lysosomal properties (LSG) and lysosomes. The protein expressed on the surface of secreting RBL cells is internalized by endocytosis via coated pits, and found in coated vesicles, endosomes, multivesicular bodies and Golgi system, before being recycled to LSG and partly delivered to lysosomes. The recycled protein is re-expressed on the surface of cells stimulated to secrete a second time.

scholarly journals Study of the transit of an integral membrane protein from secretory granules through the plasma membrane of secreting rat basophilic leukemia cells using a specific monoclonal antibody.

1986 ◽  
Vol 102 (2) ◽  
pp. 516-522 ◽  
Author(s):  
J S Bonifacino ◽  
P Perez ◽  
R D Klausner ◽  
I V Sandoval
Keyword(s):  
Monoclonal Antibody ◽  
Bovine Serum Albumin ◽  
Membrane Protein ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Secretory Granules ◽  
Integral Membrane Protein ◽  
Rat Basophilic Leukemia Cells ◽  
Rbl Cells ◽  
Basophilic Leukemia

The monoclonal antibody 5G10 reacted specifically with an 80-kD integral membrane protein in rat basophilic leukemia (RBL) cells. Immunofluorescence microscopy studies of RBL cells, fixed and permeabilized, revealed that the 80-kD protein was located in the membrane of cytoplasmic vesicles. The vesicles were identified as secretory granules by their content in immunoreactive serotonin. Expression of the 5G10 antigen on the surface of unstimulated RBL cells was low. However, RBL cells stimulated to secrete with anti-dinitrophenyl IgE followed by dinitrophenyl-bovine serum albumin or with the Ca2+ ionophore A-23187 displayed an increased expression of the antigen on their surface. Surface exposure of the 5G10 antigen was maximal at 5 min after stimulation of secretion. Removal of dinitrophenyl-bovine serum albumin from the incubation medium resulted in internalization of 50% of the antigen within 10 min.

scholarly journals VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.

1992 ◽  
Vol 118 (5) ◽  
pp. 1003-1014 ◽  
Author(s):  
T V Kurzchalia ◽  
P Dupree ◽  
R G Parton ◽  
R Kellner ◽  
H Virta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Cellular Membrane ◽  
Integral Membrane Proteins ◽  
Membrane Domains ◽  
Transport Vesicles ◽  
Vesicular Carriers ◽  
Molecular Machinery ◽  
Golgi Network

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

scholarly journals Pmel17 Initiates Premelanosome Morphogenesis within Multivesicular Bodies

2001 ◽  
Vol 12 (11) ◽  
pp. 3451-3464 ◽  
Author(s):  
Joanne F. Berson ◽  
Dawn C. Harper ◽  
Danielle Tenza ◽  
Graça Raposo ◽  
Michael S. Marks
Keyword(s):  
Membrane Protein ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Multivesicular Bodies ◽  
Posttranslational Processing ◽  
Tissue Specific ◽  
Physical Linkage ◽  
Golgi Compartment ◽  
Membrane Bound ◽  
Unknown Composition

Melanosomes are tissue-specific organelles within which melanin is synthesized and stored. The melanocyte-specific glycoprotein Pmel17 is enriched in the lumen of premelanosomes, where it associates with characteristic striations of unknown composition upon which melanin is deposited. However, Pmel17 is synthesized as an integral membrane protein. To clarify its physical linkage to premelanosomes, we analyzed the posttranslational processing of human Pmel17 in pigmented and transfected nonpigmented cells. We show that Pmel17 is cleaved in a post-Golgi compartment into two disulfide-linked subunits: a large lumenal subunit, Mα, and an integral membrane subunit, Mβ. The two subunits remain associated intracellularly, indicating that detectable Mα remains membrane bound. We have previously shown that Pmel17 accumulates on intralumenal membrane vesicles and striations of premelanosomes in pigmented cells. In transfected nonpigmented cells Pmel17 associates with the intralumenal membrane vesicles of multivesicular bodies; cells overexpressing Pmel17 also display structures resembling premelanosomal striations within these compartments. These results suggest that Pmel17 is sufficient to drive the formation of striations from within multivesicular bodies and is thus directly involved in the biogenesis of premelanosomes.

scholarly journals Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue

1999 ◽  
Vol 341 (3) ◽  
pp. 563-569 ◽  
Author(s):  
Christina WASMEIER ◽  
John C. HUTTON
Keyword(s):  
Protein Kinase ◽  
Membrane Protein ◽  
Time Course ◽  
Secretory Granules ◽  
Tyrosine Phosphatase ◽  
Signal Transduction Pathway ◽  
Integral Membrane Protein ◽  
Neuroendocrine Cells ◽  
Calmodulin Antagonist ◽  
Min6 Cells

Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic β-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K+. In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K+-stimulated phosphorylation required the presence of Ca2+. The calmodulin antagonist W-7 and the Ca2+/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the ‘inactive’ analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site.

scholarly journals Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2511-2527 ◽  
Author(s):  
F Navone ◽  
R Jahn ◽  
G Di Gioia ◽  
H Stukenbrok ◽  
P Greengard ◽  
...  
Keyword(s):  
Nervous System ◽  
Membrane Protein ◽  
Synaptic Vesicles ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Immunoblot Analysis ◽  
Integral Membrane Protein ◽  
Neuroendocrine Cells ◽  
Synapsin I ◽  
Large Dense Core Vesicles

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

scholarly journals Selection of Biophysical Methods for Characterisation of Membrane Proteins

2019 ◽  
Vol 20 (10) ◽  
pp. 2605 ◽  
Author(s):  
Tristan O. C. Kwan ◽  
Rosana Reis ◽  
Giuliano Siligardi ◽  
Rohanah Hussain ◽  
Harish Cheruvara ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Early Stage ◽  
Biological Membrane ◽  
Basic Research ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Functional Protein ◽  
Protein Research ◽  
Selection Of

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

scholarly journals Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes

1998 ◽  
Vol 274 (6) ◽  
pp. C1708-C1717 ◽  
Author(s):  
Seiji Moroi ◽  
Mitinori Saitou ◽  
Kazushi Fujimoto ◽  
Akira Sakakibara ◽  
Mikio Furuse ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Guinea Pig ◽  
Tight Junctions ◽  
Sertoli Cells ◽  
Banding Pattern ◽  
Adult Mouse ◽  
Immunofluorescence Microscopy ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Mouse Testis

Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of occludin in murine testis bearing well-developed TJ. In the adult mouse testis, occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, occludin showed a characteristic multiple banding pattern, suggesting that occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated occludin was detected. With development, occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of occludin or other TJ-associated integral membrane proteins that differ from occludin.

scholarly journals The Lumenal Domain of the Integral Membrane Protein Phogrin Mediates Targeting to Secretory Granules

Traffic ◽  
2002 ◽  
Vol 3 (9) ◽  
pp. 654-665 ◽  
Author(s):  
Christina Wasmeier ◽  
Nicholas A. Bright ◽  
John C. Hutton
Keyword(s):  
Membrane Protein ◽  
Secretory Granules ◽  
Integral Membrane Protein ◽  
Lumenal Domain

scholarly journals Monoclonal antibody HFD9 identifies a novel 28 kDa integral membrane protein on the cis-Golgi

1995 ◽  
Vol 108 (6) ◽  
pp. 2405-2414 ◽  
Author(s):  
V.N. Subramaniam ◽  
J. Krijnse-Locker ◽  
B.L. Tang ◽  
M. Ericsson ◽  
A.R. Yusoff ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Immunofluorescence Microscopy ◽  
First Integral ◽  
Integral Membrane Protein ◽  
Immunogold Labelling ◽  
Golgi Membranes ◽  
Indirect Immunofluorescence Microscopy ◽  
Golgi Region ◽  
Golgi System

We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.

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