Inhibition of phospholipase d by clathrin assembly protein 3 (AP3)

We have previously identified the presence of multiple proteins that in hibit phospatidylcholine-phospholipase I) (PLD) from rat brain rytosol (JHC. 271:1) 163-11169. 1996). Here, we purified and characterized one of the PLD inhibitory proteins from rat brain cytosol, and identified this protein as clat linn assembly protHn 3 (AIM) by peptide sequencing and immunobloi analyst AP3 hinds both inositol hexakisphosphate (IP_6) and preassemhled claihrin cages with high affinity. Iknvever. neither IP,6 binding nor clathrin cage bind ing affected the ability of AP3 lo inhibit PLD. AP3 also binds to PI( 4,5)P 2 with low affinity. But the PI(4, 5)P_2 binding was not responsible for PLD in hibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P..2. A barterially expressed fusion protein, glutiithione S-transferase-AP3 (GST-AP3). also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 ap peared lo be the result of direct interaction between AP3 and PLD because PLD bound GST AP3 in an in vitro binding assay, l'sing GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro 290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific prolein indicates that the APD dependent inhibition of PLD might play a regulatory role that is leslricti-d to the rapid eveling of synaptic vesicles.