Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles.

Fluorescence de-quenching can be used to analyze membrane lipid mixing during an in vitro fusion reaction. Here we describe a method to measure lipid mixing using vacuolar membranes purified from the yeast Saccharomyces cerevisiae. Labeling the isolated organelles with rhodamine-phosphatidylethanolamine allows to reveal ATP-dependent lipid mixing through fluorescence de-quenching in a spectrofluorometer. Combining this assay with content mixing indicators, such as the fusion-dependent maturation of a luminal vacuolar phosphatase, then permits the detection of hemifusion intermediates and the analysis of the requirements for fusion pore opening

PITPs as targets for selectively interfering with phosphoinositide signaling in cells

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase–directed strategies

Spontaneous Assembly and Induced Aggregation of Food Proteins

Beyond their nutritional value, food proteins are a versatile group of biopolymers with a considerable number of functionalities throughout their extensive structures, conformations and interaction–aggregation behaviour in solution. In the present paper, we give an overview of the induced aggregation and spontaneous reversible assembly of food proteins that lead to a diversity of supramolecular structures. After a brief description of the properties of some food proteins, the first part summarises the aggregation processes that lead to supramolecular structures with a variety of morphologies and sizes. The second part reports on the requirements that drive spontaneous assembly of oppositely charged proteins into reversible supramolecular structures. The promising new applications of these structures in food and non-food sectors are also mentioned