INHIBITION OF PHOSPHOLIPASE-D BY CLATHRIN ASSEMBLY-PROTEIN-3 (AP3)

In the accompanying paper (Chung, J,-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y, S., Morris, A, J,, and Rhee, S. G . (1997) J. Biol. Chem, 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated b y ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (P I(4,5)P-2). Here, the purification from rat brain cytosol of another P LD-inhibitory protein that is immunologically distinct horn synaptojan in is described, and this protein is identified as clathrin assembly p rotein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 bind s both inositol hexakisphosphate and preassembled clathrin cages with high affinity, However, neither inositol hexakisphosphate binding nor clathrin in cage binding affected the ability of AP3 to inhibit PLD. A P3 also binds to PT(4,5)P-2 with low affinity, But the PI(4,5)P-2 bind ing was not responsible for PLD inhibition, because the potency and ef ficacy of AP3 as an inhibitor of PLD were similar in the absence and p resence of PI(4,5)P-2. A bacterially expressed fusion protein, glutath ione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency eq ual to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins conta ining various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical far both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein i ndicates that the AP3-dependent inhibition of PLD might play a regulat ory role that is restricted to the rapid cycling of synaptic vesicles.