Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells. (49/52017)

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.  (+info)

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. (50/52017)

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.  (+info)

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons. (51/52017)

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.  (+info)

Sodium ion uptake into isolated plasma membrane vesicles: indirect effects of other ions. (52/52017)

Vesicles derived from plasma membrane of corneal endothelium were agitated to their minimum size distribution. When isotonic salt solutions surrounding the vesicles were changed there were alterations to the vesicle size distribution: the modal point of the logarithmic distribution did not change but the log variance did, indicating that substantial fission and fusion of vesicles occurred depending upon the nature of the surrounding solute. Orientation and total membrane area was conserved in the transformed population of vesicles. Although the ions added to the external isotonic salt solutions in the present series of experiments have no direct effect upon sodium membrane transporters in these membranes, kinetics of sodium accumulation into the vesicles were affected in a way that correlated with changes to the vesicle size distribution. Early-saturating (<1 min) intravesicular concentrations of sodium corresponded with apparently stable populations. Late-saturating (>1 min) intravesicular concentrations of sodium corresponded with significant vesicle distribution shifts and included a few seconds of delay. During the linear accumulation phase, both populations showed similar magnitudes of sodium transport. The significance of these data is discussed.  (+info)

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH. (53/52017)

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.  (+info)

Protein ProQ influences osmotic activation of compatible solute transporter ProP in Escherichia coli K-12. (54/52017)

ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12. Mutation proQ220::Tn5 decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP transcription or the ProP protein level. Allele proQ220::Tn5 was isolated, and the proQ sequence was determined. Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map. The proQ220::Tn5 and prc phenotypes were different, however. Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ+ bacteria but not in proQ220::Tn5 bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.  (+info)

Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. (55/52017)

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.  (+info)

Identification of mechanosensitive ion channels in the cytoplasmic membrane of Corynebacterium glutamicum. (56/52017)

Patch-clamp experiments performed on membrane fragments of Corynebacterium glutamicum fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.  (+info)