Membrane-binding proteins are key components of many signaling pathways in eukaryotic cells. Granuphilin, also known as synaptotagmin-like protein 4 (Slp-4), functions in tethering insulin secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin contains two membrane-targeting C2 domains, C2A and C2B, which interact with plasma membrane lipids including phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. Both C2A and C2B bind anionic lipids such as PI(4,5)P2 in a Ca2+-independent manner. In particular, the C2A domain binds with strong affinity to liposomes containing a physiological mixture of lipids including 24% phosphatidylserine (PS) and 2% PI(4,5)P2 (apparent Kd of 2-5 nM). The same domain binds with much weaker affinity to anionic liposomes containing either 24% PS or 2% PI(4,5)P2, but not both target lipids. Affinity is ~100-fold tighter in the presence of both target lipids, suggesting a multivalent and/or cooperative membrane binding mechanism. Here, the roles of cationic residues in two putative lipid binding pockets on the C2A domain are probed using a combination of molecular biological and biophysical approaches. Overall, granuphilin possesses several separate interactions with plasma membrane lipids and proteins, suggesting it may serve as a coincidence detector for targeting secretory vesicles to specialized regions of the plasma membrane.

Molecular basis of high-affinity membrane binding by the C2A domain of granuphilin

249th National Meeting (2015)

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