Subcellular location of phage infection protein (Pip) inLactococcus lactis

2006 ◽  
Vol 52 (7) ◽  
pp. 664-672 ◽  
Author(s):  
Duane T Mooney ◽  
Monica Jann ◽  
Bruce L Geller
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Lactococcus Lactis ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Subcellular Location ◽  
Recognition Site ◽  
Phage Infection ◽  
Sucrose Density Gradient Centrifugation ◽  
Membrane Spanning

The amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple-membrane-spanning region, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and a cell wall anchoring motif may also be present near the carboxy terminus. If functional, this recognition site could lead to covalent linkage of Pip to the cell wall. Pip was detected in both plasma membranes and envelopes (plasma membrane plus peptidoglycan) isolated from the wild-type Pip strain LM2301. Pip was firmly attached to membrane and envelope preparations and was solubilized only by treatment with detergent. Three mutant Pip proteins were separately made in which the multiple-membrane-spanning region was deleted (Pip-Δmmsr), the sortase recognition site was converted to the consensus (Pip-H841G), or the sortase recognition site was deleted (Pip-Δ6). All three mutant Pip proteins co-purified with membranes and could not be solubilized except with detergent. When membranes containing Pip-Δmmsr were sonicated and re-isolated by sucrose density gradient centrifugation, Pip-Δmmsr remained associated with the membranes. Strains that expressed Pip-H841G or Pip-Δ6 formed plaques with near unit efficiency, whereas the strain that expressed Pip-Δmmsr did not form plaques of phage c2. Both membranes and cell-free culture supernatant from the strain expressing Pip-Δmmsr inactivated phage c2. These results suggest that Pip is an integral membrane protein that is not anchored to the cell wall and that the multiple-membrane-spanning region is required for productive phage infection but not phage inactivation.Key words: phage infection protein, Pip, Lactococcus lactis, subcellular location.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

scholarly journals Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

2004 ◽  
Vol 70 (10) ◽  
pp. 5825-5832 ◽  
Author(s):  
Kitt Dupont ◽  
Thomas Janzen ◽  
Finn Kvist Vogensen ◽  
Jytte Josephsen ◽  
Birgitte Stuer-Lauridsen
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Receptor Binding ◽  
Insertional Mutagenesis ◽  
Binding Proteins ◽  
Phage Infection ◽  
Terminal Part ◽  
Cell Wall Polysaccharides ◽  
Phage Adsorption ◽  
Membrane Spanning

ABSTRACT The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and φ645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that φ645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that φ645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and φ645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and φ645 that showed no homology in the C-terminal part.

ATTACHMENT TO THE LUTEAL PLASMA MEMBRANES: AN EARLY EVENT IN THE ACTION OF LUTEINIZING HORMONE

1973 ◽  
Vol 57 (2) ◽  
pp. 199-NP ◽  
Author(s):  
HANNU RAJANIEMI ◽  
TAPANI VANHA-PERTTULA
Keyword(s):  
Plasma Membrane ◽  
Luteinizing Hormone ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Early Event ◽  
Target Tissue ◽  
Marker Enzyme ◽  
Sucrose Density Gradient Centrifugation ◽  
Initial Event

SUMMARY The distribution of 125I-labelled luteinizing hormone (LH) was studied by autoradiography of the murine pregnant ovary 10 min after intravenous administration. The grains in autoradiograms were localized around the luteal cells. The pregnant ovary showed the highest uptake of 125I-labelled LH 30 min after the injection. No similar accumulation of 125I-labelled follicle-stimulating hormone, 125I-labelled albumin or free 125I was obtained. The ribosomal fraction of the corpus luteum contained a slightly higher level of radioactivity than the other subcellular fractions after injection of 125I-labelled LH. Sucrose density gradient centrifugation of the luteal particle preparation resulted in an enrichment of radioactivity in particles containing Na+,K+-activated ATPase, a marker enzyme of the plasma membrane. These findings support the concept of plasma membrane binding as the initial event in LH action on the target tissue.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line

2004 ◽  
Vol 32 (5) ◽  
pp. 777-779
Author(s):  
P. Pompach ◽  
P. Man ◽  
P. Novák ◽  
V. Havlíček ◽  
A. Fišerová ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Extraction Procedure ◽  
Sucrose Density Gradient ◽  
Cellular Adhesion ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Sucrose Density Gradient Centrifugation ◽  
Biologically Relevant ◽  
Jurkat T Cell

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

scholarly journals The αβ monomer of the insulin receptor has hormone-responsive tyrosine kinase activity

1991 ◽  
Vol 273 (1) ◽  
pp. 49-56 ◽  
Author(s):  
E R Mortensen ◽  
J G Drachman ◽  
G Guidotti
Keyword(s):  
Plasma Membranes ◽  
Kinase Activity ◽  
Gradient Centrifugation ◽  
Insulin Receptors ◽  
Sucrose Density Gradient ◽  
Erythrocyte Membranes ◽  
Protein Kinase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Adipocyte Plasma Membranes ◽  
Receptor Dimers

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema

2002 ◽  
Vol 282 (6) ◽  
pp. L1382-L1390 ◽  
Author(s):  
Paola Palestini ◽  
Chiara Calvi ◽  
Elena Conforti ◽  
Laura Botto ◽  
Carla Fenoglio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Activation ◽  
Membrane Lipids ◽  
Gradient Centrifugation ◽  
Mechanical Stimuli ◽  
Biophysical Properties ◽  
Interstitial Edema ◽  
Vascular Perfusion

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml · kg−1 · min−1 saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (∼4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

scholarly journals Up against the Wall: Is Yeast Cell Wall Integrity Ensured by Mechanosensing in Plasma Membrane Microdomains?

2014 ◽  
Vol 81 (3) ◽  
pp. 806-811 ◽  
Author(s):  
Christian Kock ◽  
Yves F. Dufrêne ◽  
Jürgen J. Heinisch
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Yeast Cell ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Yeast Cell Wall ◽  
Membrane Microdomains ◽  
Wall Structure ◽  
Fungal Cell ◽  
Membrane Spanning

ABSTRACTYeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

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