Inhibition of phospholipase D by amphiphysins

Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brai n cytosol were previously purified and identified as synaptojanin and AP180 , which are specific to nerve terminals and associate with the clathrin coa t. Two additional PLD-inhibitory proteins have now been purified and identi fied as the amphiphysins I and II, which forms a heterodimer that also asso ciates with the clathrin coat. Bacterially expressed recombinant amphiphysi ns inhibited both PLD1 and PLDS isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of similar to 1 5 nM). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2, Coprecipita tion experiments suggested that the inhibitory effect of amphiphysins resul ts from their direct interaction with PLDs. The NH2 terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic aci d formed by signal-induced PLD is thought to be required for the assembly o f clathrin-coated vesicles during endocytosis, Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in sy naptic vesicle trafficking.