Background: PI(4,5)P 2 -and tyrosine phosphorylation-dependent unconventional secretion of FGF2 is mediated by direct translocation across the plasma membrane. Results: PI(4,5)P 2 -mediated membrane recruitment causes oligomerization of tyrosine-phosphorylated FGF2 that, in turn, triggers the formation of a lipidic membrane pore. Conclusion: Membrane-inserted FGF2 oligomers represent intermediates of membrane translocation during unconventional secretion. Significance: Mechanistic insight into a novel self-sustained mechanism of protein translocation across membranes is provided.

Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgiindependent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P 2 is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P 2 is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P 2 resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P 2 that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P 2 associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS Ist2 ) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS Ist2 targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P 2 at the PM created ER-PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca 2+ entry (SOCE). The CSS Ist2 and the C-terminus of the ER-located Ca 2+ sensor STIM2 were sufficient to bind PI(4,5)P 2 and PI(3,4,5)P 3 at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P 2 , suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.-Temmerman, K., and W. Nickel. A novel flow cytometric assay to quantify interactions between proteins and membrane lipids.

Bacteria living in a competitive environment are able to secrete proteinaceous toxins, known as bacteriocins, which can kill closely related bacterial competitors while causing little harm to the bacteriocinogenic cells. These bacterial inhibitors are produced by all major groups of Bacteria (53). Bacteriocin production has also been reported for Halobacteriaceae, a family of extremely halophilic Archaea (36). Bacteriocins constitute a structurally and functionally diverse group within the antimicrobials, ranging from small peptides, such as microcins of Enterobacteriaceae and antibiotics secreted by low-GC-content gram-positive bacteria (23, 31), to middle-sized polypeptides, such as colicins of Escherichia coli (34) and their counterparts in Pseudomonas aeruginosa (S pyocins) (39), to large phage tail-like multiprotein complexes, such as syringacin produced by Pseudomonas syringae (58) and R-and F-type pyocins of P. aeruginosa (39). Bacteriocin mode of killing can be either by membrane pore formation, nonspecific degradation of cellular DNA, cleavage of 16S rRNA or tRNA, or inhibition of peptidoglycan synthesis resulting in cell lysis (51).Bacteriocins from lactic acid bacteria, such as nisin, have received considerable attention, given their potential as food preservatives (41, 42). Among bacteriocins of gram-negative bacteria, colicins are the most intensively studied. Colicins are produced by, and attack, strains of E. coli. These proteins, as well as the S pyocins, are organized in functional domains, namely, regions for cell attachment, translocation, and bactericidal activity. Colicins and S pyocins parasitize specific membrane-bound receptors on target cells, resulting in a rather narrow spectrum of activity. The host strain is protected from its own bacteriocin through the action of a cognate immunity protein that is coproduced with the bacteriocin (39, 51).It has been proposed that bacteriocins may play a key role in bacterial population dynamics (52,53). In a recent study, Kirkup and Riley demonstrated that the production of colicins by E. coli colonizing the mouse colon gives the colicinogenic strains a competitive advantage by killing colicin-sensitive E. coli sharing the same ecological niche (33). Possible applications of bacteriocinogenic strains in agriculture include their use in biological control of soilborne or phyllosphere-inhabiting bacterial plant pathogens. In this way, heterologous production of the peptide bacteriocin trifolitoxin by an avirulent Agrobacterium strain effectively enhances biological control of Agrobacterium vitis crown gall (24). Expression of the trifolitoxin genes from Rhizobium leguminosarum bv. trifolii T24 in Rhizobium etli CE3 increases bean nodulation competitiveness of the recombinant strain in the presence of indigenous rhizobia under agricultural conditions (54). A phage tail-like bacteriocin (serracin P) produced by Serratia plymithicum may be used for biological control of fire blight caused by Erwinia amylovora (30), while Xanthomonas campestris pv. glycin...