Influenza neuraminidase is delivered directly to the apical surface of MDCK cell monolayers

AbstractThe aim of this study was to investigate whether influenza neuraminidase travels directly from the Golgi complex to the apical domain of the plasma membrane in virally infected epithelial (MDCK) cell monolayers, or whether it passes transiently through the basolateral domain. Using a new assay for the delivery of neuraminidase to the plasma membrane, we found that the time course of transport of this protein from the Golgi complex to the apical surface of MDCK cell monolayers was very similar to that for influenza haemagglutinin, which is known to be delivered directly to its destination. In addition, a similar time course of neuraminidase transport was found in BHK cells, which are not asymmetric and in which delivery must therefore be direct. Finally, basolateral exposure of MDCK cell monolayers grown on nitro-cellulose filters to an anti-neuraminidase antibody was shown to have no effect on the delivery of active neuraminidase to the apical surface. We conclude from these results that neuraminidase, like haemagglutinin, is delivered directly to the apical surface

Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy

AbstractBacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA

Membrane fusion: All done with SNAREpins?

AbstractSNARE proteins are sufficient to fuse artificial membranes together. In the cell, vesicle transport may rely on fusion mediated by interaction between vesicle (v) and target (t) SNAREs, whereas the homotypic fusion of organelle biogenesis may be mediated by t-SNARE–t-SNARE interaction

Transport of influenza virus envelope proteins from the Golgi complex to the apical plasma membrane in MDCK cells: pH-Controlled interaction with a cycling receptor is not involved

AbstractIn influenza virus-infected monolayers of the epithelial cell line MDCK the viral envelope proteins, haemagglutinin and neuraminidase, are targetted specifically to the apical surface. In this study we have tested the hypothesis that the polarized delivery of these proteins to the plasma membrane involves the operation of a receptor that cycles between the trans Golgi network and the plasma membrane, binding the proteins at low pH in the former compartment and releasing them at normal extracellular pH in the latter. The hypothesis predicts that apical, but not basolateral, low pH would eventually delay or block delivery of the proteins to the plasma membrane. We found that basolateral low pH in fact had the more profound effect, in line with its greater effect on intracellular pH. We conclude that the hypothesis is not valid, and that low extracellular pH causes its effect on protein transport by changing intracellular pH

Characteristics of a cell-free assay for the delivery of proteins to the plasma membrane

AbstractWe have previously described the reconstitution, in a cell-free system, of the constitutive delivery of a newly synthesized protein, influenza neuraminidase, to the plasma membrane in BHK cells. Here we report some of the characteristics of this in vitro membrane fusion event. We show that fusion requires ATP hydrolysis, and exploit this requirement to distinguish the time-course of fusion from that of neuraminidase action. In addition, we present evidence for the occurrence of multiple fusions between hybrid membrane vesicles

Synthetic peptides of the rab3 effector domain stimulate a membrane fusion event involved in regulated exocytosis

AbstractWe have developed a system in which the fusion of pancreatic zymogen granules with plasma membranes can be studied in vitro. Here we show that this membrane fusion event is stimulated specifically by peptides of the effector domain of rab3, a small, monomeric GTP-binding protein. In addition, we demonstrate that the stimulatory effect of the peptides involves their binding to a target on the plasma membrane, and is both qualitatively and quantitatively different from the effect of GTPγS, which also enhances membrane fusion. We suggest that regulated exocytosis in the pancreatic acinar cell may be under the control of more than one type of GTP-binding protein

Two modes of secretion in pancreatic acinar cells: Involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca2+ entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. The part played by Ca2+ released from intracellular stores in the regulation of secretion is well established; however, the role of Ca2+ influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (≥10 μM) elicited an additional component of exocytosis despite reducing Ca2+ influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (∼70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 μM) was potentiated (∼45%) by wortmannin. Exocytosis stimulated by 1 μM acetylcholine in the absence of extracellular Ca2+ was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca2+ entry through store-operated Ca2+ channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd3+ (2 μM), but not by inhibition of noncapacitative Ca2+ entry by SB203580 (10 μM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca2+ influx