scholarly journals Lateral diffusion of wild-type and mutant Ld antigens in L cells.

1984 ◽  
Vol 99 (6) ◽  
pp. 2333-2335 ◽  
Author(s):  
M Edidin ◽  
M Zuniga
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Histocompatibility Antigen ◽  
Wild Type ◽  
Major Histocompatibility Antigen ◽  
Cytoplasmic Side

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

scholarly journals Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

2019 ◽  
Author(s):  
Wai Cheng (Christine) Wong ◽  
Jz-Yuan Juo ◽  
Yi-Hung Liao ◽  
Ching-Ya Cheng ◽  
Chih-Hsiang Lin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Protein Dynamics ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Cell Plasma

AbstractNative cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

scholarly journals Role of Cytoplasmic Domain Serines in Intracellular Trafficking of Furin

2004 ◽  
Vol 15 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Florencia B. Schapiro ◽  
Thwe Thwe Soe ◽  
William G. Mallet ◽  
Frederick R. Maxfield
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Endocytic Recycling ◽  
Late Endosomes

Furin is a transmembrane protein that cycles between the plasma membrane, endosomes, and the trans-Golgi network, maintaining a predominant distribution in the latter. It has been shown previously that Tac-furin, a chimeric protein expressing the extracellular and transmembrane domains of the interleukin-2 receptor α chain (Tac) and the cytoplasmic domain of furin, is delivered from the plasma membrane to the TGN through late endosomes, bypassing the endocytic recycling compartment. Tac-furin also recycles in a loop between the TGN and late endosomes. Localization of furin to the TGN is modulated by a six-amino acid acidic cluster that contains two phosphorylatable serines (SDSEED). We investigated the role of these serines in the trafficking of Tac-furin by using a mutant chimera in which the SDS sequence was replaced by the nonphosphorylatable sequence ADA (Tac-furin/ADA). Although the mutant construct is internalized and delivered to the TGN, both the postendocytic trafficking and the steady-state distribution were found to differ from the wild-type. In contrast with Tac-furin, Tac-furin/ADA does not enter late endosomes after being internalized. Instead, it traffics with transferrin to the endocytic recycling compartment, and from there it is delivered to the TGN. As with Tac-furin, Tac-furin/ADA is sorted from the TGN into late endosomes at steady state, but its retrieval from the late endosomes to the TGN is inhibited. These results suggest that serine phosphorylation plays an important role in at least two steps of Tac-furin trafficking, acting as an active sorting signal that mediates the selective sorting of Tac-furin into late endosomes after internalization, as well as its retrieval from late endosomes back to the TGN.

scholarly journals Signal transduction by glycophorin A: role of extracellular and cytoplasmic domains in a modulatable process.

1988 ◽  
Vol 107 (4) ◽  
pp. 1351-1357 ◽  
Author(s):  
J A Chasis ◽  
M E Reid ◽  
R H Jensen ◽  
N Mohandas
Keyword(s):  
Signal Transduction ◽  
Transmembrane Protein ◽  
Fold Increase ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Glycophorin A ◽  
Domain Antibody ◽  
Extracellular Region ◽  
Cytoplasmic Side

Binding of ligands to the extracellular region of the erythrocyte transmembrane protein glycophorin A induces a decrease in membrane deformability. Since the property of membrane deformability is regulated by the skeletal proteins on the cytoplasmic side of the membrane, this suggests that ligand binding may initiate a transmembrane signal. To further study this process, we examined which domains of the extracellular region of glycophorin are involved in signal transduction and whether the cytoplasmic domain of the molecule is necessary for transmitting the signal. Using the ektacytometer, we compared the effect on deformability of four monoclonal antibodies that detect different epitopes on glycophorin A. We found that 9A3 (which recognized the amino terminus of glycophorin) caused a 5.8-fold increase in rigidity, R-10 and 10F7 (which recognized epitopes in the mid-region of the extracellular domain) caused a 10.8-fold increase in rigidity and B14 (which binds to glycophorin close to the membrane) caused a 18-fold increase in rigidity. Further, a direct relationship was observed between the degree of antibody-induced rigidity and the amount of glycophorin A that became associated with the skeletal proteins in a Triton shell assay. In Miltenberger V erythrocytes, which contain a hybrid sialoglycoprotein with no cytoplasmic domain, antibody binding did not induce an increase in rigidity. These results imply that glycophorin A is capable of a modulatable form of transmembrane signaling that is determined by the extracellular domain to which the ligand binds, and the cytoplasmic domain of glycophorin A is crucial for this process.

scholarly journals Rat ovarian lutropin receptor is a transmembrane protein. Evidence for an Mr-20,000 cytoplasmic domain

1986 ◽  
Vol 239 (1) ◽  
pp. 83-87 ◽  
Author(s):  
K P Keinänen ◽  
H J Rajaniemi
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Membrane Vesicles ◽  
Cytoplasmic Domain ◽  
Proteolytic Digestion ◽  
Plasma Membrane Vesicles ◽  
Human Choriogonadotropin ◽  
Lutropin Receptor ◽  
Membrane Bound ◽  
Inside Out

Membrane topography of the rat ovarian lutropin receptor was studied by two different approaches. Ovarian membrane preparation, labelled with 125I-labelled human choriogonadotropin in vivo, was subjected to extensive chymotryptic digestion. The soluble and membrane-bound radioactive complexes were cross-linked with glutaraldehyde, and analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. Chymotrypsin solubilized 70-75% of the radioactivity as Mr-96,000, Mr-74,000 and Mr-61,000 complexes, and decreased the size of the membrane-bound 125I-labelled human choriogonadotropin-receptor complex from Mr 130,000 to Mr 110,000. The Mr-110,000 complex was not observed when 0.1% Triton X-100 was present in the proteolytic digestion. Enrichment of inside-out-oriented plasma-membrane vesicles by concanavalin A affinity chromatography increased by 70% the fraction of radioactivity that remained in the membrane fraction after chymotrypsin treatment. Chymotrypsin also diminished the size of the membrane-bound unoccupied receptor from Mr 90,000 to Mr 70,000, as detected by ligand (125I-labelled human choriogonadotropin) blotting. These results suggest that the lutropin receptor is a transmembrane protein with a cytoplasmic domain of Mr 20,000 that is sensitive to proteolytic digestion in the inside-out-oriented plasma-membrane vesicles.

scholarly journals Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

1986 ◽  
Vol 103 (2) ◽  
pp. 327-331 ◽  
Author(s):  
E Livneh ◽  
M Benveniste ◽  
R Prywes ◽  
S Felder ◽  
Z Kam ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Diffusion Coefficients ◽  
Egf Receptor ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Protein Kinase Activity ◽  
Lateral Mobility ◽  
Cos Cells ◽  
Epidermal Growth

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

scholarly journals Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains.

1989 ◽  
Vol 109 (6) ◽  
pp. 3325-3331 ◽  
Author(s):  
W F Wade ◽  
J H Freed ◽  
M Edidin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Major Histocompatibility Complex ◽  
Cytoplasmic Domain ◽  
Translational Diffusion ◽  
Type I ◽  
Beta Chain ◽  
Wild Type ◽  
Alpha Chain ◽  
Histocompatibility Complex

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.

scholarly journals Polarized Insertion of New Membrane from a Cytoplasmic Reservoir during Cleavage of the Drosophila Embryo

2000 ◽  
Vol 150 (4) ◽  
pp. 849-860 ◽  
Author(s):  
Thomas Lecuit ◽  
Eric Wieschaus
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Protein ◽  
Lateral Diffusion ◽  
Drosophila Embryo ◽  
Membrane Insertion ◽  
Membrane Growth ◽  
Specialized Form ◽  
Membrane Reservoir ◽  
Polarized Epithelium

Cellularization of the Drosophila embryo is a specialized form of cytokinesis that results in the formation of a polarized epithelium. The mechanisms of membrane growth during cytokinesis are largely unknown. It is also unclear whether membrane growth and polarization represent distinct processes that occur simultaneously or whether growth of the membrane is involved in the emergence of polarity. Using a combination of surface labeling and particles tracking techniques, we monitored the dynamics of marked membrane regions during cellularization. We find that the major source of membrane is intracellular, rather than in the form of a plasma membrane reservoir. Depolymerization of microtubules inhibits the export of a newly synthesized transmembrane protein from the Golgi apparatus to the plasma membrane and simultaneously blocks membrane growth. Membrane insertion occurs in a defined sequence at specific sites, first apical, then apical–lateral. Diffusion of the membrane appears insufficient to compete with the massive local insertion of new membrane. We thus identify a tightly regulated scheme of polarized membrane insertion during cellularization. We propose that such a mechanism could participate in the progressive emergence of apical–basal polarity.

scholarly journals Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids.

1993 ◽  
Vol 120 (1) ◽  
pp. 25-35 ◽  
Author(s):  
G M Lee ◽  
F Zhang ◽  
A Ishihara ◽  
C L McNeil ◽  
K A Jacobson
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Colloidal Gold ◽  
Plasma Membranes ◽  
Membrane Surface ◽  
Mean Square Displacement ◽  
Lateral Diffusion ◽  
Pericellular Matrix ◽  
Cultured Fibroblasts ◽  
Average Diffusion

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.

scholarly journals Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2607-2618 ◽  
Author(s):  
L M Roman ◽  
H Garoff
Keyword(s):  
Semliki Forest Virus ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Directed Transport ◽  
Membrane Spanning ◽  
Darby Canine Kidney ◽  
Canine Kidney

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild-type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.

scholarly journals The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein

2005 ◽  
Vol 168 (6) ◽  
pp. 855-862 ◽  
Author(s):  
Rose E. Goodchild ◽  
William T. Dauer
Keyword(s):  
Endoplasmic Reticulum ◽  
Glutamic Acid ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Wild Type ◽  
Interacting Protein ◽  
Dyt1 Dystonia ◽  
Conserved Domain ◽  
Lumenal Domain ◽  
Aaa Protein

A glutamic acid deletion (ΔE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the ΔE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of “substrate trap” EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.

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