scholarly journals Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

2007 ◽  
Vol 408 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Gabriele Zanchetti ◽  
Paolo Colombi ◽  
Marta Manzoni ◽  
Luigi Anastasia ◽  
Luigi Caimi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Surface Protein ◽  
Experimental Conditions ◽  
Cellular Processes ◽  
Endosomal Compartment ◽  
Outer Leaflet ◽  
Peripheral Membrane Protein ◽  
The Moment

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.

scholarly journals Plasma Membrane Proton ATPase Pma1p Requires Raft Association for Surface Delivery in Yeast

2001 ◽  
Vol 12 (12) ◽  
pp. 4129-4138 ◽  
Author(s):  
Michel Bagnat ◽  
Amy Chang ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Triton X100 ◽  
Proton Atpase ◽  
Peripheral Membrane Protein ◽  
And Function ◽  
Cellular Compartments

Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments. In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface. Disruption of rafts led to mistargeting of Pma1p to the vacuole. Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association. One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7. Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer. We suggest that clustering facilitates partition of Pma1p into rafts and transport to the cell surface.

scholarly journals Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

1991 ◽  
Vol 115 (5) ◽  
pp. 1357-1374 ◽  
Author(s):  
L S Musil ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Surface Protein ◽  
Immunohistochemical Localization ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional ◽  
Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

scholarly journals Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells.

1987 ◽  
Vol 35 (8) ◽  
pp. 809-816 ◽  
Author(s):  
R Pakkanen ◽  
K Hedman ◽  
O Turunen ◽  
T Wahlström ◽  
A Vaheri
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Apical Cell ◽  
Antigenic Site ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
Choriocarcinoma Cells

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.

scholarly journals A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes.

1991 ◽  
Vol 115 (2) ◽  
pp. 505-515 ◽  
Author(s):  
A Corlu ◽  
B Kneip ◽  
C Lhadi ◽  
G Leray ◽  
D Glaise ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Surface Protein ◽  
Cell Aggregation ◽  
Cell Interaction ◽  
Cell Contact ◽  
Plasma Membrane Protein ◽  
Extracellular Matrix Components

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.

Lipoxygenase Forms Located at the Plant Plasma Membrane

1995 ◽  
Vol 50 (1-2) ◽  
pp. 29-36 ◽  
Author(s):  
Arndt Nellen ◽  
Barbara Rojahn ◽  
Helmut Kindl
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lipid Body ◽  
Integral Membrane Protein ◽  
Long Distance ◽  
Two Phase ◽  
Cucumis Sativus L ◽  
Sds Page ◽  
Peripheral Membrane Protein ◽  
Two Phase Partition

Abstract In cucumber cotyledons (Cucumis sativus L.) containing several soluble and particulate forms of lipoxygenase (LOX), the location of LOX forms in microsomes has been studied. We concentrated on the question whether the plasma membrane contains one or more forms of LOX. As methodology, we applied both the two-phase partition with polyethylene glycol/dextran and density gradient flotation of plasma membrane-enriched membrane fractions. Both methods show that a high percentage of the microsomal LOX can be attributed to the plasma membrane. Emphasis was put on the findings that the LOX located at the plasma membrane consisted of a species behaving as an integral membrane protein and another form characterized as a peripheral membrane protein by solubilization with carbonate. With long distance SDS-PAGE and immunodecoration using anti-lipid body LOX antiserum, it is possible to distinguish between microsomal LOX forms by small but significant differences in size. Treatment of seedlings with methyl jasmonate led to an enhanced level of LOX at the plasma membrane.

scholarly journals Elimination of defective alpha-factor pheromone receptors.

1997 ◽  
Vol 17 (11) ◽  
pp. 6236-6245 ◽  
Author(s):  
D D Jenness ◽  
Y Li ◽  
C Tipper ◽  
P Spatrick
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Half Life ◽  
Structural Defects ◽  
Receptor Protein ◽  
Mutant Form ◽  
Wild Type ◽  
Alpha Factor ◽  
Mutant Cells

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane. When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C.

scholarly journals Extracellular pH Dynamically Controls Cell Surface Delivery of Functional TRPV5 Channels

2006 ◽  
Vol 27 (4) ◽  
pp. 1486-1494 ◽  
Author(s):  
Tim T. Lambers ◽  
Elena Oancea ◽  
Theun de Groot ◽  
Catalin N. Topala ◽  
Joost G. Hoenderop ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Surface Protein ◽  
Receptor Potential ◽  
Transport Processes ◽  
Extracellular Ph ◽  
Cell Surface Expression ◽  
Transient Receptor Potential Vanilloid

ABSTRACT Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca2+-selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca2+-transporting cells, contributing significantly to the overall Ca2+ balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, 45Ca2+ uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular “kiss and linger” interactions with the plasma membrane.

scholarly journals Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

2017 ◽  
Vol 216 (10) ◽  
pp. 3275-3290 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Membrane ◽  
Physiological Role ◽  
Amino Acid Transporters ◽  
Genetic Dissection ◽  
Cellular Processes ◽  
Rag Gtpases ◽  
Endosomal Recycling ◽  
Recycling Pathway

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface.

scholarly journals Type II Phosphatidylinositol 4-Kinase β Is a Cytosolic and Peripheral Membrane Protein That Is Recruited to the Plasma Membrane and Activated by Rac-GTP

2002 ◽  
Vol 277 (48) ◽  
pp. 46586-46593 ◽  
Author(s):  
Yong Jie Wei ◽  
Hui Qiao Sun ◽  
Masaya Yamamoto ◽  
Pawel Wlodarski ◽  
Kaiko Kunii ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Type Ii ◽  
Peripheral Membrane Protein ◽  
Phosphatidylinositol 4
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