scholarly journals Intracellular localization of the P21rho proteins.

1992 ◽  
Vol 119 (3) ◽  
pp. 617-627 ◽  
Author(s):  
P Adamson ◽  
H F Paterson ◽  
A Hall
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Biological Function ◽  
Intracellular Localization ◽  
Heterologous Proteins ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Early Endosomes ◽  
Chimeric Molecules ◽  
Palmitoylation Site

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

Intracellular Localization Of F.VIII R:Ag In Platelet And Endothelial Cells Using Peroxidase Fab Conjugates In Electron Microscopy

1981 ◽  
Author(s):  
C Jeanneau ◽  
Y Sultan
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Cellular Localization ◽  
Culture Medium ◽  
Intracellular Localization ◽  
Fab Fragments ◽  
Thrombin Activation ◽  
Storage Pool Disease ◽  
Papain Digestion ◽  
Specific Rabbit

In previous studies (Jeanneau et al Thromb. Haemost.1977, 38, 42) specific rabbit antibodies against human F.VIII R:Ag isolated by immuno absorption on insolubilized polymers from monospecific rabbit antisera against the F.VIII/ VW factor molecule and coupled to peroxidase showed the labelling of platelet and endothelial cell membranes but did not allow to visualize intra cellular localization of this antigen. Preparation of the peroxidase coupled Fab fragments of the same specific antibodies showed the intra cellular localization of F.VIII R:Ag in platelet and endothelial cells. Fab antibody fragments were prepared using papain digestion. Fab and Fc fragments were separated from the non digested antibody by filtration through Sephedex G 100. The Fab fragments were then separated from the Fc using CM 52 carboxymethyl cellulose.In human washed platelets, staining was observed on the plasma membrane, the canalicular system and some granules. After thrombin activation the release of granules containing F.VIII R:Ag better visualized in the canalicular system. In patients with storage pool disease only some platelets showed normal SCS labelling, however no stained granules were observed.Native human endothelial cells were obtained from umbilical cord veins, washed and resuspended in culture medium prior to incubation with peroxidase Fab conjugates. After fixation in glutaraldehyde and exposure to diaminobenzidine, the peroxidase staining was seen on the plasma membrane and in a large number of vacuoles. In some endothelial cells the Golgi apparatus appeared labelled demonstrating evidence of F.VIII R:Ag synthesis by endothelial cells.

scholarly journals Calcium- and proton-dependent relocation of annexin A6 in Jurkat T cells stimulated for interleukin-2 secretion.

2007 ◽  
Vol 54 (2) ◽  
pp. 261-271 ◽  
Author(s):  
Paulina Podszywalow-Bartnicka ◽  
Agnieszka Strzelecka-Kiliszek ◽  
Joanna Bandorowicz-Pikula ◽  
Slawomir Pikula
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Interleukin 2 ◽  
Jurkat T Cells ◽  
Vesicular Traffic ◽  
Early Endosomes ◽  
Intracellular Ca ◽  
Stimulation Of

Annexin A6 (AnxA6) is a Ca(2+)-dependent membrane-binding protein involved in vesicular traffic. The likely participation of AnxA6 in the response of lymphocytes to Ca(2+) signals has not been investigated yet. The present study focuses on intracellular relocation of AnxA6 in human Jurkat T lymphoblasts upon stimulation followed by transient increase of intracellular [Ca(2+)] and exocytosis of interleukin-2 (IL-2). Stimulation of the cells under different experimental conditions (by lowering pH and/or by rising extracellular [Ca(2+)] in the presence of ionomycin) induced time-dependent transients of intracellular [Ca(2+)] and concomitant changes in AnxA6 intracellular localization and in IL-2 secretion, with only minor effects on cell viability and apoptosis. In resting conditions (in the presence of EGTA or with no ionophore) AnxA6 was localized uniformly in the cytosol, whereas it translocated to vesicular structures beneath the plasma membrane within 5 min following stimulation of Jurkat T cells and rise of intracellular [Ca(2+)] at pH 7.4. Lowering the extracellular pH value from 7.4 to 6.0 significantly enhanced this process. AnxA6 changed its location from the cytosol to the secretory granules and early endosomes which seem to represent membranous targets for annexin. In conclusion, AnxA6 is sensitive to variations in intracellular [Ca(2+)] upon stimulation of Jurkat T cells, as manifested by a switch in its intracellular localization from the cytosol to vesicular structures located in close proximity to the plasma membrane, suggestive of participation of AnxA6 in calcium- and proton-dependent secretion of cytokines by lymphocytes.

scholarly journals Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2

2011 ◽  
Vol 301 (1) ◽  
pp. F226-F235 ◽  
Author(s):  
Lene Stødkilde ◽  
Rikke Nørregaard ◽  
Robert A. Fenton ◽  
Guixian Wang ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Ureteral Obstruction ◽  
Cellular Localization ◽  
Collecting Duct ◽  
Intracellular Localization ◽  
Water Channel ◽  
Degradation Pathway ◽  
Salt Transport ◽  
Apical Surface ◽  
Altered Expression ◽  
Early Endosomes

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.

scholarly journals Brain I3 Binding Protein regulates K-Ras4B membrane localization and signaling

2021 ◽  
Author(s):  
Ian McCabe ◽  
Huanqing Zhang ◽  
Jonathan A. Cooper ◽  
David L. Turner ◽  
Anne B. Vojtek
Keyword(s):  
Plasma Membrane ◽  
Biological Function ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Membrane Localization ◽  
Biological Functions ◽  
Ras Proteins ◽  
Binding Partner ◽  
Endosomal Compartment ◽  
Oncogenic Activity

Membrane localization of Ras proteins is necessary for their biological functions and oncogenic activity. We report here on the identification of Brain I3 Binding Protein (BRI3BP) as a novel binding partner for Ras. We show that K-Ras4B plasma membrane localization and biological function are reduced in the absence of BRI3BP. BRI3BP interacts with K-Ras4B and K-Ras4A and our data suggest that BRI3BP operates within the recycling endosomal compartment to regulate K-Ras localization to the plasma membrane. This study uncovers a new regulatory protein for Ras membrane localization.

scholarly journals TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in Arabidopsis thaliana

2018 ◽  
Author(s):  
Akira Yoshinari ◽  
Barbara Korbei ◽  
Junpei Takano
Keyword(s):  
Arabidopsis Thaliana ◽  
Plasma Membrane ◽  
Intracellular Localization ◽  
The Other ◽  
Dependent Manner ◽  
Root Cells ◽  
Polar Localization ◽  
Other Hand ◽  
Early Endosomes ◽  
Vacuolar Sorting

ABSTRACTBoron (B) is an essential micronutrient for plants, however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of boron (B) toward the root stele in Arabidopsis thaliana. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. To avoid over-accumulation of B, BOR1 in the plasma membrane is rapidly internalized and transported into the vacuole for proteolysis after high-B supply in an ubiquitination-dependent manner. Although BOR1 has been predicted to be transported into multi-vesicular bodies/late endosomes (MVB/LEs) via the endosomal sorting complex required for transport (ESCRT) machinery, experimental evidence was absent so far. In this study, we investigated the intracellular localization of BOR1 by visualizing endomembrane compartments, and tested the involvement of ESCRT-0-like proteins TOM1-LIKEs (TOLs) in the vacuolar sorting of BOR1. Under low-B conditions, a large portion of cytoplasmic BOR1 was localized in the trans-Golgi networks/early endosomes (TGN/EEs) labeled with VHA-a1 subunit. Pharmacological interference of endosomal recycling using brefeldin A induced colocalization of BOR1 with RabA5D, which labels recycling vesicles associated with the TGN. These data suggest that BOR1 cycles between plasma membrane and TGN/EE via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, under high-B conditions, BOR1 was localized in the inside of TOL5-positive MVB/LEs. To examine the roles of TOL proteins in intracellular trafficking of BOR1, we analyzed BOR1-GFP localization in the TOL quintuple mutant (tolQ; tol2-1tol3-1tol5-1tol6-1tol9-1) after high-B supply. In the tolQ mutant, vacuolar sorting of BOR1 was delayed, while the polar localization of BOR1 was not disturbed. Taken together, BOR1 is constantly transported to the TGN/EE by endocytosis and recycled to the plasma membrane likely via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, BOR1 is transported to the vacuole via TOL5-positive MVB/LEs under high-B conditions. TOL proteins are required for sorting of ubiquitinated BOR1 into MVB/LE for vacuolar degradation but not for the polar trafficking of BOR1.

scholarly journals Endocytosis and the Src family of non-receptor tyrosine kinases

2014 ◽  
Vol 5 (2) ◽  
pp. 143-155 ◽  
Author(s):  
James Reinecke ◽  
Steve Caplan
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Tyrosine Kinases ◽  
Intracellular Transport ◽  
Intracellular Localization ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
Related Family ◽  
Spatiotemporal Regulation ◽  
Early Endosomes

AbstractThe regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to, internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane through tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src) and its closely related family members Yes and Fyn represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. As Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases.

scholarly journals NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane

2021 ◽  
Vol 4 (5) ◽  
pp. e202000972
Author(s):  
Ian M Ahearn ◽  
Helen R Court ◽  
Farid Siddiqui ◽  
Daniel Abankwa ◽  
Mark R Philips
Keyword(s):  
Plasma Membrane ◽  
Ras Proteins ◽  
The Third ◽  
Carboxyl Methylation ◽  
Hypervariable Regions ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Direct Delivery ◽  
Protein Chaperone ◽  
Palmitoylation Site

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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