Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment

Abstract

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Lipid-associated PML domains regulate CCTα, Lipin1 and lipid homeostasis

“…PA, which promotes Lipin1 association with membranes (Ren et al, 2010), was not detected on nLDs or cLDs with a biosensor suggesting it is below the detection threshold or the biosensor is not sufficiently PA-specific, as was suggested previously (Horchani et al, 2014). Lipin1a was not detected on cLDs and associated with accumulation of DAG on cLDs in PML KO cells, which could reflect a partial block in TAG synthesis or increased TAG hydrolysis.…”

The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

“…GFP fusion proteins based on Spo20 51-91 have been established (Nakanishi, 2004) and optimized (Kassas et al, 2012;Zhang et al, 2014) to act as biosensors for PA in living cells. More recent studies by Antonny and coworkers reported that Spo20 does not efficiently distinguish between the anionic lipids, PA, PS and PIPs (Horchani et al, 2014). Instead, they found that the membrane binding of Spo20 51-91 relies on the negative charge density on the surface of the liposomes.…”

The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain

“…The Q-SNARE Spo20 contains an amphipathic helix with a polybasic face that binds PA on prospores and the plasma membrane as a positive regulator of the protein (42). Its binding to PA depends on the presence of the acidic lipids PS and PI(4,5)P 2 (43). In mammalian cells syntaxin1 contains a PBR that binds to various PIs on lipid overlay experiments, whereas liposome binding shows that it preferentially binds to PA (44).…”