Faculty Opinions recommendation of Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane.

2017 ◽  
Author(s):  
David Cafiso
Keyword(s):  
Plasma Membrane ◽  
Membrane Attachment

scholarly journals Actin-membrane interaction in fibroblasts: what proteins are involved in this association?

1984 ◽  
Vol 99 (1) ◽  
pp. 95s-103s ◽  
Author(s):  
P Mangeat ◽  
K Burridge
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Stress Fiber ◽  
Membrane Interaction ◽  
Microfilament Bundles ◽  
The Family ◽  
Form Part ◽  
Membrane Attachment ◽  
A Chain ◽  
And Function

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.

Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells

1993 ◽  
Vol 105 (3) ◽  
pp. 613-628 ◽  
Author(s):  
T. David-Pfeuty ◽  
S. Bagrodia ◽  
D. Shalloway
Keyword(s):  
Plasma Membrane ◽  
Nuclear Translocation ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Nuclear Staining ◽  
Membrane Breakdown ◽  
Membrane Attachment ◽  
Transforming Activity ◽  
Main Components ◽  
Nih 3T3

Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both normal, myristoylated pp60c-src and a nonmyristoylated mutant, pp60c-src/myr-, has been used to compare the effects of preventing myristoylation on the localization of c-Src in NIH 3T3-derived overexpresser cells using immunofluorescence microscopy. During interphase, pp60c-src partitions between the plasma membrane and the centrosome, while pp60c-src/myr- is predominantly cytoplasmic but also partly nuclear. The cytoplasmic, but not the nuclear, staining can be readily washed out by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr-, in contrast with the nuclear one, does not associate tightly with structures that are insoluble in the presence of nonionic detergents. We have previously shown that during G2 phase, pp60c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that pp60c-src/myr- translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr-, but not of pp60c-src, is also observed when cells are arrested in G0 or at the G1/S transition. Furthermore, during mitosis, pp60c-src is found primarily in diffuse and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr- more specifically associates with the main components of the spindle apparatus (poles and fibers) and inside the interchromosomal space. These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts are readily moved into the nucleus. They also raise the possibility that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.

scholarly journals N-Terminal Motifs in Some Plant Disease Resistance Proteins Function in Membrane Attachment and Contribute to Disease Resistance

2012 ◽  
Vol 25 (3) ◽  
pp. 379-392 ◽  
Author(s):  
Daigo Takemoto ◽  
Maryam Rafiqi ◽  
Ursula Hurley ◽  
Greg J. Lawrence ◽  
Maud Bernoux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Disease Resistance ◽  
Golgi Apparatus ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Protein Accumulation ◽  
Resistance Proteins ◽  
Membrane Attachment ◽  
Signal Anchor ◽  
Terminal Domains

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

scholarly journals Two Distinct Low-pH Steps Promote Entry of Vaccinia Virus

2007 ◽  
Vol 81 (16) ◽  
pp. 8613-8620 ◽  
Author(s):  
Alan C. Townsley ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Low Ph ◽  
Surface Proteins ◽  
Step Model ◽  
Endosomal Acidification ◽  
Membrane Attachment ◽  
Endosomal Pathway ◽  
Rate Limiting ◽  
Virion Surface

ABSTRACT Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37°C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.

GCET2, a New Adaptor Protein in B Cell Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1917-1917
Author(s):  
Zenggang Pan ◽  
Yulei Shen ◽  
Baosheng Ge ◽  
Cheng Du ◽  
Timothy McKeithan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Germinal Center ◽  
Adaptor Protein ◽  
Phosphorylation Sites ◽  
Germinal Center B Cell ◽  
Membrane Attachment ◽  
Cloned Gene

Abstract GCET2 (Germinal Center B-cell Expressed Transcript 2; also named HGAL, Human Germinal center-Associated Lymphoma) is a newly cloned gene that has been shown to be a useful marker for germinal center (GC) B-cells and GC B-cell derived malignancies, including follicular lymphomas and germinal center B cell-like diffuse large B-cell lymphomas (GCB-DLBCLs). GCET2 is also a prognostic indicator for DLBCLs, and patients with higher expression of GCET2 have significantly better survival than those with lower expression. We cloned GCET2 from a number of GC B cell-restricted expressed sequence tags (ESTs) in order to study the differentiation of GC B-cells and to elucidate the mechanisms underlying the GC reaction, which is not well understood. We are report here the biochemical and biological properties of GCET2, which may help to determine its role in the GC reaction. Sequence analysis of GCET2 did not reveal any known domains but predicted five tyrosine phosphorylation sites, all of which are conserved in its mouse homologue, M17, also a GC B cell-restricted transcript. We first determined the cellular localization of GCET2 using subcellular fractionation of a B cell line, DHL16, and found that GCET2 is constitutively localized in cellular membranes but is excluded from lipid rafts. These findings were further confirmed by fluorescence confocal microscopy. GCET2 does not have a transmembrane domain but has a putative myristoylation site and a putative palmitoylation site, which may mediate its membrane attachment. Using 3H metabolic labeling, we demonstrated that GCET2 was both myristoylated and palmitoylated, and GCET2 lost its membrane association after mutating both of these sites, indicating that the membrane attachment of GCET2 is mediated by these two post-translational modifications. We then studied tyrosine phosphorylation of GCET2. In both Daudi and DHL16 cells, GCET2 was phosphorylated following pervanadate treatment. By serially mutating the five predicated tyrosine-phosphorylation sites, we found that the distal three sites are crucial for GCET2 phosphorylation. GCET2 was also phosphorylated when co-transfected into COS7 cells with protein tyrosine kinases (PTKs) LYN, LCK or SYK, and therefore it may be a substrate of these kinases in B cells. GCET2 has a conserved GRB2 binding site, and it indeed associates with GRB2 following pervanadate treatment. Our data suggest that GCET2 acts as an adaptor protein in GC B-cells by transducing signals from GC B-cell membrane to the cytosol. Our working model is as follows: a stimulus to GC B-cells induces the activation of PTKs LYN and SYK, which sequentially phosphorylate GCET2 at the plasma membrane. Phosphorylated GCET2 then recruits GRB2 from the cytosol to the plasma membrane, and this complex further recruits additional partners and activates downstream pathways, which function in the GC reaction. We are currently identifying other proteins in the GCET2/GRB2 complex to determine the pathways downstream of GCET2 activation.

scholarly journals THE SELECTIVE INHIBITION OF MACROPHAGE PHAGOCYTIC RECEPTORS BY ANTI-MEMBRANE ANTIBODIES

1972 ◽  
Vol 135 (3) ◽  
pp. 458-475 ◽  
Author(s):  
Phillip Holland ◽  
Nancy H Holland ◽  
Zanvil A. Cohn
Keyword(s):  
Plasma Membrane ◽  
Selective Inhibition ◽  
Morphological Evidence ◽  
Igg Antibodies ◽  
High Concentration ◽  
Mouse Macrophages ◽  
Γ Globulins ◽  
Membrane Attachment ◽  
Common Antigens ◽  
Partial Blockade

Rabbit antibodies were prepared against purified mouse macrophages, erythrocytes, and liver lysosomes. In the presence of complement each of these reagents was capable of lysing mouse erythrocytes and macrophages. In the absence of complement, all antisera agglutinated mouse erythrocytes and at high concentration produced a cytotoxic effect on macrophages. At IgG concentrations of 100 µg/ml, no morphological evidence of cytotoxicity was evident. These data suggest the presence of common antigens on the erythrocyte and macrophage plasma membrane. Anti-macrophage, anti-erythrocyte, and anti-lysosomal γ-globulins and IgG, employed at subtoxic concentrations, all inhibited the attachment and ingestion of opsonized erythrocytes and mycoplasma. This occurred without significant reduction in the phagocytosis of polystyrene particles, formalinized erythrocytes, and yeast cell walls. Each of the anti-membrane IgG antibodies was capable of blocking the Fc receptor on the macrophage plasma membrane. Attachment to the macrophage membrane occurred by means of the Fab region. However, a role for the Fc portion of the molecule was suggested since pepsin-digested IgG was unable to block the receptor. Each of the IgG antibodies produced a partial blockade of the complement receptor and reduced the ingestion of EAC1,4,2,3 by approximately 50%.

scholarly journals Plasma Membrane Localization of Gαz Requires Two Signals

1998 ◽  
Vol 9 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Janine Morales ◽  
C. Simone Fishburn ◽  
Paul T. Wilson ◽  
Henry R. Bourne
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Protein Localization ◽  
Receptor Activation ◽  
Α Subunit ◽  
Surface Receptors ◽  
Defective Mutant ◽  
Specific Localization ◽  
Membrane Attachment ◽  
Stable Association

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

scholarly journals A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.

1987 ◽  
Vol 105 (2) ◽  
pp. 819-831 ◽  
Author(s):  
A A Rogalski
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Cultured Cells ◽  
Molecular Forms ◽  
Cell Coupling ◽  
Membrane Structures ◽  
Adult Chicken ◽  
Attachment Sites ◽  
Membrane Attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.

scholarly journals Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells

1988 ◽  
Vol 256 (1) ◽  
pp. 103-108 ◽  
Author(s):  
F Fouchier ◽  
P Bastiani ◽  
T Baltz ◽  
D Aunis ◽  
G Rougon
Keyword(s):  
Staphylococcus Aureus ◽  
Plasma Membrane ◽  
Phospholipase C ◽  
Chromaffin Cells ◽  
Enzyme Concentration ◽  
Glycosylphosphatidylinositol Anchor ◽  
Membrane Attachment ◽  
Molecular Masses

Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms.

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