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.
Highlights
The identity of cellular membranes results from the combination of specific determinants such as rare phosphoinositides and small G proteins [1, 2], and bulk physicochemical properties [3, 4]
Better characterizing the lipid binding properties of the Spo20 membrane sensor region is crucial considering that probes based on this sequence are becoming increasingly popular to follow the production of phosphatidic acid (PA) in cells [15, 36, 37]
We have found no evidence for a stereospecific interaction between PA and the Spo20 membrane sensor region
Summary
The identity of cellular membranes results from the combination of specific determinants such as rare phosphoinositides and small G proteins [1, 2], and bulk physicochemical properties [3, 4]. Because the Kennedy pathway occurs at the endoplasmic reticulum whereas PLDs are recruited to other organelles and notably the plasma membrane, different pools of PA probably coexist in the cell [12]. Tracking these pools is difficult and there is clearly a need for better readouts of PA in a cellular context [14]; the interest for protein regions that have been reported to sense PA [15, 16, 17]. There is no consensus domain or sequence for the general recognition of PA by proteins
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