Synaptojanin Inhibition of Phospholipase D Activity by Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate

Abstract

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.

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

“…Such degradation is likely to be catalyzed to a significant extent by synaptojanin 1, a major polyphosphoinositide phosphatase in brain. Synaptojanin 1 dephosphorylates PtdIns(4,5)P 2 and PtdIns (4)P via its inositol 5-phosphatase domain and Sac1 domain, respectively (16,17,46,47). It therefore is thought to represent a negative regulator of membrane-clathrin coat interactions.…”

“…PLD is inhibited by endocytic proteins such as synaptojanin, AP180, and amphiphysin. Although AP180 and amphiphysin directly interact with PLD (50,51), the inhibition of PLD by synaptojanin is attributed to its ability to dephosphorylate PtdIns(4,5)P 2 (20,46), which is a cofactor for PLD (51).…”

Phosphoinositides in Constitutive Membrane Traffic

“…One group of these enzymes contains only a NH 2 -terminal Sac domain, and the second class contains an additional, 5-phosphatase domain in the center of the protein followed by various other domains (155). Sac1p was identified in a screen for mutations that relieved the block in secretion caused by loss of activity of the S. cerevisiae PITP, Sec14p (51,385), and was subsequently discovered to have PIP phosphatase activity (47,124). In S. cerevisiae there are two such proteins, Sac1p and Fig4p, and in humans three, KIAA0274, KIAA0966, and KIAA0851.…”

Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes

“…However, it is unknown whether these events occur at a pre-or postsynaptic location. Furthermore, the potential control of PLD at a presynaptic location would be of particular interest as it has recently been reported that synucleins (Jenco et al, 1998) and synaptojanin (Chung et al, 1997), proteins located abundantly at the presynaptic terminal, can act as endogenous PLD inhibitors, implying that PLD action at the presynaptic terminal is physiologically signi®cant. The aim of our study was to examine the signal transduction pathway which links mGluRs to PLD in our presynaptic-rich rat cerebrocortical synaptosome preparation and to determine whether PKC activation, occurring secondary to PLC activation, is involved in the activation process.…”