Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Extracellular ATP and adenosine modulate tumor necrosis factor-induced lysis of L929 cells in the presence of actinomycin D

Extracellular ATP in concentrations of 0.5 to 2.5 mM modulates TNF-induced cytolysis of L929 cells in the presence of actinomycin D. When present throughout the entire assay period, it inhibits the TNF-induced cytolysis. ADP was less active whereas AMP and GTP were nonreactive. However, inhibition was also achieved by adenosine that was nearly as active as ATP. Yet, the inhibitory effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine. Thus, the nonhydrolyzable ATP analogue adenyl(beta-gamma-methylendiphosphate) was equally effective in inhibiting TNF-induced cytolysis. Moreover, no conversion of ATP into adenosine was observed during the entire assay period. However, inhibition no longer occurred when the TNF and ATP containing medium was removed after 5 h and replaced by a fresh medium containing TNF and no ATP. We now observed substantial enhancement of the TNF-induced cytolysis by ATP. Finally, treatment with N6-(R-phenylisopropyl)adenosine or with aminophylline, which are thought to downregulate adenosine receptors and to prevent binding of ligands to adenosine receptors, respectively, abolishes adenosine and ATP-mediated inhibition. Again, substantial enhancement of the TNF-induced cytolysis was observed by ATP and only a minor effect by adenosine. The results together suggest that ATP interacts with purinoceptors on the plasma membrane and is capable to enhance and inhibit TNF-induced cytolysis under appropriate conditions. The outcome of the ATP-induced modulation may be influenced by adenosine receptors.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.