The effects of phospholipids and fatty acids on the oligomer formation of NAP-22

Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM fraction of the neuronal cell membrane. Previous studies showed tight binding activity of NAP-22 to acidic membrane lipids and the self-interaction of NAP-22, i.e., oligomerization. In this study, the effect of various phospholipids, lysophospholipids and fatty acids on the oligomerization of NAP-22 was studied through SDS-PAGE after chemical cross-linking and electron microscopic observation. High molecular mass oligomers were detected by SDS-PAGE after incubation in solutions containing over 20 mM NaCl at pH 6.5-8.5, even in the absence of lipid addition, and the addition of Ca²⁺/calmodulin abolished oligomerization. Higher molecular mass oligomer formation after incubation with acidic phospholipids was detected with gradient SDS-PAGE. Much higher mass oligomers were detected in the presence of polyunsaturated fatty acids. Electron microscopic analysis of the samples after SDS treatment showed tangled rope-like structures. Liposome-bound NAP-22 showed small oval or annular structures after cross-linking and SDS treatment. These oligomers were suggested to make the tangled rope-like structures, for annular structures of the same size were observed in the structure. These results suggest the participation of NAP-22 to liquid-liquid phase separation through oligomerization.