Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin

1996 ◽  
Vol 109 (6) ◽  
pp. 1285-1295
Author(s):  
F. Le Caherec ◽  
P. Bron ◽  
J.M. Verbavatz ◽  
A. Garret ◽  
G. Morel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Digestive Tract ◽  
Xenopus Oocytes ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Plasma Membrane Proteins ◽  
Cicadella Viridis

Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 × 10(−3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 × 10(−3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

2007 ◽  
Vol 292 (3) ◽  
pp. C1132-C1136 ◽  
Author(s):  
Alexandre Leduc-Nadeau ◽  
Karim Lahjouji ◽  
Pierre Bissonnette ◽  
Jean-Yves Lapointe ◽  
Daniel G. Bichet
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Xenopus Laevis ◽  
Western Blot ◽  
Plasma Membranes ◽  
Transient Receptor Potential ◽  
Expression System ◽  
Vitelline Membrane ◽  
Transient Receptor Potential Vanilloid ◽  
Xenopus Laevis Oocytes

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na+-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins

1980 ◽  
Vol 58 (10) ◽  
pp. 1156-1164 ◽  
Author(s):  
Paul C. Holland ◽  
George A. Cates ◽  
Byron S. Wenger ◽  
Barbara L. Raney
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Protein Biosynthesis ◽  
Sodium Dodecyl ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Plasma Membrane Proteins ◽  
Dystrophic Muscle

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate – polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

1988 ◽  
Vol 66 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Jon G. Church ◽  
Shobha Ghosh ◽  
Basil D. Roufogalis ◽  
Antonio Villalobo
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Line ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Normal Adult ◽  
Plasma Membrane Proteins ◽  
Phosphoprotein Phosphatase ◽  
Membrane Bound

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [γ-32P]ATP or [γ-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform–methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.

Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1667-1676 ◽  
Author(s):  
A. Mayer ◽  
I.E. Ivanov ◽  
D. Gravotta ◽  
M. Adesnik ◽  
D.D. Sabatini
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Great Promise ◽  
Plasma Membrane Proteins ◽  
In Vitro System ◽  
Viral Glycoproteins ◽  
Basolateral Plasma Membrane

An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15–30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.

scholarly journals Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach

1983 ◽  
Vol 210 (1) ◽  
pp. 37-47 ◽  
Author(s):  
B T Pan ◽  
R Blostein ◽  
R M Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Plasma Membrane Proteins ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Cell Plasma ◽  
A Subunit

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

275 EVIDENCE FOR α5 AND αV INTEGRINS ON SPERM PLASMA MEMBRANES AND THEIR ROLE IN BOVINE FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 294
Author(s):  
R. F. Gonçalves ◽  
R. P. Bertolla ◽  
V. H. Barnabe
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Weighted Least Squares ◽  
Gradient Centrifugation ◽  
Plasma Membrane Proteins ◽  
Western Blots ◽  
Integrin Αv ◽  
Sds Page

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of α5 and αV integrins on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to these integrin subunits. To determine if integrin subunits were present on sperm, sperm plasma membrane proteins were subjected to 1-dimensional SDS-PAGE and Western blot analysis. Frozen-thawed sperm, donated by ABS Pecplan, were centrifuged at 700 × g for 10 min, washed twice with warm PBS (Nutricell®, Campinas, Sao Paulo, Brazil), and resuspended in Jones buffer (0.4% deoxyclolic acid, 8.9 M sucrose, 0.1 M Tris, pH 8.5) for 60 min at 4°C to solubilize sperm plasma membranes. Plasma membrane proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The resulting blots were probed with αV integrin antibody (Calbiochem®, San Diego, CA, USA) or α5 integrin antibody (Calbiochem) and developed using ECL. Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with anti-integrin αV IgG (2), and FM with anti-integrin a5 IgG (3). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 1 × 105 washed, pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Each experiment was repeated 4 times and data from each experiment were pooled. Approximately 80 to 90 oocytes per treatment for fertilization were evaluated in each replicate. Weighted least squares means were used to analyze fertilization data (SAS software, SAS Institute, Cary, NC, USA). The significance level for all tests was P < 0.05. Both antibodies for α5 (35 kDa) and αV (34 kDa) integrins showed immunoreactivity on Western blots of sperm membrane proteins. Addition of anti-integrin αV, and anti-integrin α5 decreased fertilization (P < 0.05) compared with the control: (1) 94.1 ± 1.0%; (2) 18.2 ± 1.0%; (3) 12.2 ± 1.0%. These findings show that αV and α5 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization. This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.

scholarly journals Separate Mechanisms for the Inhibition of Platelet Aggregation by Adenosine and 2-Cloroadenosine

1977 ◽  
Author(s):  
R. Apitz-Castro ◽  
C.R. Torres
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Electrophoretic Pattern ◽  
Membrane Component ◽  
Plasma Membrane Proteins ◽  
Adp Receptor ◽  
Selective Increase ◽  
Specific Membrane

The mechanism by which adenosine (Ado) and 2-cloroadenosine (Cl-Ado) inhibit platelet aggregation is not clear. In order to get some insight into the mode of action of these compounds, we studied the effect of Cl-Ado on the uptake of Ado by intact platelets, the effect of these compounds on the endogenous phosphorylation of specific plasma membrane proteins, and its effect on the carboxymethylation pattern of plasma membrane proteins in intact platelets. Cl-Ado does not modify the uptake of Ado by intact platelets, nor is itself incorporated into the platelet’s pool of nucleotides. Phosphorylation of plasma membrane proteins is not affected by Cl-Ado; however, Ado produces a selective increase in the phosphorylation of one plasma membrane component of glycoproteic nature. As has been reported, phosphorylation of this glycoprotein is also modulated by cAMP (BBA, 455:371, 1976). Although the electrophoretic pattern of carboxymethylated plasma membranes is unaffected by Ado or Cl-Ado, it was found that the former markedly increases the label of all the susceptible proteins, while Cl-Ado selectively protects a single membrane component. Electrophoretically, this component seems to be related to the above mentioned glycoprotein. The results reported suggest that Ado and Cl-Ado interact with different components of the plasma membrane, impairing platelet aggregation through different mechanisms. In the case of Ado, two ways seem operative: a) A cAMP-like stimulation of a specific membrane glycoprotein and b) A more general perturbation of the membrane structure, perhaps through an Ado-carrier complex (Acta Med. Scand. 525:169, 1971). Cl-Ado seems to interact solely on the external surface of the plasma membrane, suggesting that the transmembrane phospho-glycoprotein previously described is in some way closely related to the ADP-receptor of the platelet plasma membrane.

scholarly journals The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

2020 ◽  
Vol 21 (20) ◽  
pp. 7573
Author(s):  
Satish Kantipudi ◽  
Jean-Marc Jeckelmann ◽  
Zöhre Ucurum ◽  
Patrick D. Bosshart ◽  
Dimitrios Fotiadis
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Heavy Chain ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Light Chains ◽  
Substrate Affinity ◽  
Amino Acid Transporters

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes

2000 ◽  
Vol 440 (4) ◽  
pp. 627-633 ◽  
Author(s):  
D. Singer-Lahat ◽  
N. Dascal ◽  
L. Mittelman ◽  
S. Peleg ◽  
I. Lotan
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Xenopus Oocytes ◽  
Plasma Membrane Proteins
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