A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P 3 ) and that it does not catalyze the dephosphorylation of either I(1,3,4)P 3 or inositol 1,4-bisphosphate. However, synaptojanin hydrolyzes both the 4-and 5-phosphates of PI(4,5)P 2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P 3 , but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P 2 . Synaptojanin did not inhibit PLD in the absence of PI(4,5)P 2 , and the extent of PLD inhibition was related to the extent of PI(4,5)P 2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P 2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P 2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P 2 , facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P 2 , and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P 2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.
Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brain cytosol were previously purified and identified as synaptojanin and AP180, which are specific to nerve terminals and associate with the clathrin coat. Two additional PLD-inhibitory proteins have now been purified and identified as the amphiphysins I and II, which forms a heterodimer that also associates with the clathrin coat. Bacterially expressed recombinant amphiphysins inhibited both PLD1 and PLD2 isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of ϳ15 nM). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2. Coprecipitation experiments suggested that the inhibitory effect of amphiphysins results from their direct interaction with PLDs. The NH 2 terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic acid formed by signal-induced PLD is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in synaptic vesicle trafficking.
15980 -15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to 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 inhibition, 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 bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal 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 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 protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.We recently showed that rat brain cytosol contains proteins that inhibit the activity of partially purified brain membrane phospholipase D (PLD) 1 stimulated by ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) (1). Sequential chromatography of the brain cytosolic preparation yielded four inhibitory fractions, which exhibited differential sensitivity to heat. The most heat-labile inhibitor (inhibitor IA) was purified to yield a 150-kDa protein, which, in the accompanying paper (2), we showed to be synaptojanin, a nerve terminal-enriched protein with inositol polyphosphate phosphatase (IPPase) activity, and which inhibits PLD by hydrolyzing PI(4,5)P 2 .We now describe the purification of PLD inhibitor IB, which has an apparent molecular mass of 165 kDa and is less susceptible to heat treatment than synaptojanin. Sequences of tryptic peptides of inhibitor IB suggest that it is identical to clathrin assembly protein 3 (AP3). We further show that AP3 binds to PLD. EXPERIMENTAL PROCEDURESMaterials-The sources of rat brains, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), GTP␥S, PI(4,5)P 2 , [cholinemethyl-3 H]dipalmitoylphosphatidylcholine ((pam) 2 PC) (50 Ci/mmol), [2-palmitoyl-9,10-3 H](pam) 2 PC (89 Ci/mmol), and n-octyl--D-glucopyranoside were as described previously (2). Phytic acid (inositol hexakisphosphate, or IP 6 ) and monoclonal antibodies to AP3 (clone AP180-I) were obtained from Sigma. PLD, AR...
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