scholarly journals Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b

1998 ◽  
Vol 330 (2) ◽  
pp. 1015-1021 ◽  
Author(s):  
Gunnar BUYSE ◽  
Dominique TROUET ◽  
Thomas VOETS ◽  
Ludwig MISSIAEN ◽  
Guy DROOGMANS ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Chloride Channel ◽  
Mammalian Cells ◽  
Chloride Channels ◽  
Subcellular Location ◽  
Structural Similarity ◽  
Confocal Imaging ◽  
Intracellular Location ◽  
Intracellular Chloride

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

A double-label confocal imaging method for detecting the internalization of bacteria by non-phagocytic mammalian cells

1994 ◽  
Vol 52 ◽  
pp. 216-217
Author(s):  
M. H. Chestnut ◽  
L. L. Odioso ◽  
T. E. Otte ◽  
B.C. Hulette
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Confocal Imaging ◽  
Imaging Method ◽  
Thin Sections ◽  
Double Label ◽  
Human Gingival Epithelial Cells ◽  
Immunofluorescence Labeling

Internalization by non-phagocytic mammalian cells is a key part of the life cycle of several important pathogenic bacteria. Mechanistic studies of this event often include transmission electron micrographs intended to demonstrate the presence of bacteria within the mammalian cell, but these data are often uncompelling. It is very difficult to be certain, based on the appearance and number of phospholipid membranes surrounding the bacterial cell, if it is enclosed in a vesicle, is surrounded directly by mammalian cell cytoplasm, or is merely contained within an invagination of the plasma membrane. Serial thin sections could provide the needed clarification, but are almost never presented, presumably because of the effort involved. Here we offer an alternative method, combining immunofluorescence labeling of the bacterium and fluorescence staining of the mammalian cell plasma membrane with a lipophilic dye, to visualize the internalization of bacteria by mammalian cells.Freshly isolated human gingival epithelial cells were cultured in keratinocyle serum-free medium (KSFM). Second passage cells were seeded on coverslips and placed in 24-well plates.

scholarly journals Sarco/endoplasmic-reticulum calcium ATPase SERCA1 is maintained in the endoplasmic reticulum by a retrieval signal located between residues 1 and 211

2003 ◽  
Vol 371 (3) ◽  
pp. 775-782 ◽  
Author(s):  
Thomas NEWTON ◽  
John P. J. BLACK ◽  
John BUTLER ◽  
Anthony G. LEE ◽  
John CHAD ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Subcellular Location ◽  
Terminal Segment ◽  
Calcium Atpase ◽  
Endoplasmic Reticulum Calcium ◽  
Calcium Pumps ◽  
Green Fluorescent ◽  
Fast Twitch

The location of sarco/endoplasmic-reticulum calcium ATPase (SERCA) retention/retrieval motifs in the sequence of the SERCA1 has been investigated by examining the subcellular location in COS-7 cells of enhanced-green-fluorescent-protein-tagged calcium-pump chimaeras. These chimaeras have been constructed from the fast-twitch SERCA1 and the plasma-membrane calcium ATPase PMCA3. The N-terminal, central and C-terminal segments of these calcium pumps were exchanged between SERCA1 and PMCA3. The segments exchanged correspond to residues 1–211, 212–711 and 712–994 of SERCA1, and residues 1–264, 265–788 and 789–1159 of PMCA3 respectively. Only chimaeras containing the N-terminal segment of SERCA1 were located in the endoplasmic reticulum (ER), whereas chimaeras containing the N-terminal segment from PMCA3 were able to escape from the ER and enter the endomembrane pathway en route for the plasma membrane. Co-localization of SERCA1 in COS-7 cells with the ER/Golgi-intermediate compartment marker ERGIC53 indicates that SERCA1 is maintained in the ER by a process of retrieval. These results indicate that the N-terminal region of SERCA1, containing transmembrane helices M1 and M2, contains an ER-retrieval signal.

scholarly journals Identification of TMEM206 proteins as pore of ASOR acid-sensitive chloride channels

2019 ◽  
Author(s):  
Florian Ullrich ◽  
Sandy Blin ◽  
Katina Lazarow ◽  
Tony Daubitz ◽  
Jens-Peter von Kries ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Ion Channels ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Ion Permeation ◽  
Anion Channels ◽  
Acid Sensing Ion Channels ◽  
Genome Wide ◽  
A Genome

ABSTRACTAcid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated anion channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying ASOR chloride channel. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR’s pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR’s role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.

scholarly journals Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

2013 ◽  
Vol 24 (17) ◽  
pp. 2703-2713 ◽  
Author(s):  
Philip D. Fox ◽  
Christopher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Jenny L. Higgins ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Surface Proteins ◽  
Hek 293 ◽  
Cortical Endoplasmic Reticulum

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

Some Contributions of Phosphatase and 3,3'-Diaminobenzidine Cytochemistry to Cell Biology

1977 ◽  
Vol 35 ◽  
pp. 420-423
Author(s):  
Alex B. Novikoff
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Structure And Function ◽  
Cell Organelles ◽  
Thiamine Pyrophosphatase ◽  
The Status ◽  
Dab Cytochemistry ◽  
And Function

This presentation will highlight cytochemical studies that have illuminated some aspects of structure and function of the endoplasmic reticulum (ER); these have been reviewed recently (1, 2). Phosphatase (Pase) cytochemistry has led to the formulation of new questions regarding secretory mechanisms in a number of endocrine and exocrine cells; it has also made the status of GERL as a distinct organelle considerably firmer. 3,31-diaminobenzidine (DAB) cytochemistry has revealed the presence of an organelle apparently ubiquitous in mammalian cells, the anucleoid peroxisomes(microperoxisomes). DAB cytochemistry has been utilized recently by Gonatas et al. (3) to demonstrate that internalized plasma membrane is transported to GERL.Our initial use of Pase cytochemistry to visualize cell organelles included nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase), and acid Pase (AcPase). NDPase hydrolyzes the diphosphates of inosine, uridine, and guanosine but not cytidine or adenine diphosphates. Since the work of Yamasaku and Hayaishi (4) we have used TPPase and NDPase interchangeably.

Mammalian GPI-anchor modifications and the enzymes involved

2020 ◽  
Vol 48 (3) ◽  
pp. 1129-1138 ◽  
Author(s):  
Yi-Shi Liu ◽  
Morihisa Fujita
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Mammalian Cells ◽  
Gpi Anchor ◽  
C Terminus ◽  
Lipid Moiety ◽  
Human Proteins

Glycosylphosphatidylinositol (GPI) is a glycolipid added to the C-terminus of a large variety of proteins in eukaryotes, thereby anchoring these proteins to the cell surface. More than 150 different human proteins are modified with GPI, and GPI-anchored proteins (GPI-APs) play critical roles in embryogenesis, neurogenesis, immunity, and fertilization. GPI-APs are biosynthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the Golgi apparatus. During transport, GPI-APs undergo structural remodeling that is important for the efficient folding and sorting of GPI-APs. Asparagine-linked glycan-dependent folding and deacylation by PGAP1 work together to ensure that correctly folded GPI-APs are transported from the ER to the Golgi. Remodeling of the GPI lipid moiety is critical for the association of GPI-APs with lipid rafts. On the cell surface, certain GPI-APs are cleaved by GPI cleavage enzymes and released from the membrane, a key event in processes such as spermatogenesis and neurogenesis. In this review, we discuss the enzymes involved in GPI-AP biosynthesis and the fate of GPI-APs in mammalian cells, with a focus on the assembly, folding, degradation, and cleavage of GPI-APs.

scholarly journals Full-Length, Glycosylated NSP4 Is Localized to Plasma Membrane Caveolae by a Novel Raft Isolation Technique

2007 ◽  
Vol 81 (11) ◽  
pp. 5472-5483 ◽  
Author(s):  
Stephen M. Storey ◽  
Thomas F. Gibbons ◽  
Cecelia V. Williams ◽  
Rebecca D. Parr ◽  
Friedhelm Schroeder ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Mdck Cells ◽  
Resonance Energy ◽  
Full Length ◽  
Confocal Imaging ◽  
Isolation Technique ◽  
Caveolin 1 ◽  
Specific Subset ◽  
Golgi Network

ABSTRACT Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.

scholarly journals The C-terminal Dilysine Motif for Targeting to the Endoplasmic Reticulum Is Not Required for Cf-9 Function

2001 ◽  
Vol 14 (3) ◽  
pp. 412-415 ◽  
Author(s):  
Renier A. L. Van der Hoorn ◽  
Anke Van der Ploeg ◽  
Pierre J. G. M. de Wit ◽  
Matthieu H. A. J. Joosten
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Resistance Gene ◽  
Subcellular Location ◽  
Cladosporium Fulvum ◽  
Specific Recognition

The tomato resistance gene Cf-9 encodes a membrane-anchored, receptor-like protein that mediates specific recognition of the extracellular elicitor protein AVR9 of Cladosporium fulvum. The C-terminal dilysine motif (KKRY) of Cf-9 suggests that the protein resides in the endoplasmic reticulum. Previously, two conflicting reports on the subcellular location of Cf-9 were published. Here we show that the AARY mutant version of Cf-9 is still functional in mediating AVR9 recognition, suggesting that functional Cf-9 resides in the plasma membrane. The data presented here and in reports by others can be explained by masking the dilysine signal of Cf-9 with other proteins.

scholarly journals Plasma Membrane CFTR Regulates RANTES Expression via Its C-Terminal PDZ-Interacting Motif

2003 ◽  
Vol 23 (2) ◽  
pp. 594-606 ◽  
Author(s):  
Kim Estell ◽  
Gavin Braunstein ◽  
Torry Tucker ◽  
Karoly Varga ◽  
James F. Collawn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Inflammatory Diseases ◽  
Airway Epithelial Cells ◽  
Dominant Negative ◽  
Cystic Fibrosis Gene ◽  
Channel Activity ◽  
Signaling Complex

ABSTRACT Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTR's chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane.

scholarly journals Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

2015 ◽  
Vol 112 (16) ◽  
pp. E2004-E2013 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Yasunori Saheki ◽  
Pietro De Camilli
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Lipid Binding ◽  
Dense Layer ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

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