Inhibitory effect of NAP-22 on the phosphatase activity of synaptojanin-1

Endocytosis of the synaptic vesicle is a complicated process, in which many proteins and lipids participate. Phosphatidylinositol 4,5‐bisphosphate (PIP2) plays important roles in the process, and the dynamic regulation of this lipid is one of the key events. Synaptojanin is a PIP2 phosphatase, and dephosphorylation of PIP2 of the clathrin coated‐vesicle results in the uncoating of the vesicle. NAP‐22 is one of the major proteins of the neuronal detergent‐resistant membrane microdomain and localizes in both the presynaptic plasma membrane and the synaptic vesicle. To elucidate the role of NAP‐22 in synaptic function, a screening of the NAP‐22 binding proteins through pull‐down assay was performed. In addition to CapZ protein, synaptojanin‐1 was detected by LC‐MS/MS, and Western blotting using antisynaptojanin‐1 confirmed this result. The interaction seems to be important in the course of synaptic vesicle endocytosis, because NAP‐22 inhibited the phosphatase activity of synaptojanin in a dose‐dependent manner. The inhibitory region for 5‐phosphatase and the binding region for PIP2 overlapped in the amino acid sequence of NAP‐22, so elucidation of the regulatory mechanism of the PIP2 binding ability of NAP‐22 could be important in understanding the membrane dynamics at the presynaptic region. © 2011 Wiley Periodicals, Inc.