Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins.

Abstract

Abstract. Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.S YNAPSIN I is a neuron-specific synaptic vesicle-associated phosphoprotein (11,13,20,32,35). It has a collagenase-insensitive head domain containing a serine residue that can be phosphorylated by cAMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase I (site 1) and an elongated collagenase-sensitive tail domain with two serine residues that can be phosphorylated by calcium/calmodulin-dependent protein kinase II (sites 2 and 3) (10,19,31,39). The association of synapsin I with highly purified brain small synaptic vesicles has recently been partially characterized (34). Purified dephosphorylated synapsin I bound to synapsin I-depleted synaptic vesicles with high affinity (K, = 10 nM at 40 mM NaC1) and saturability (Bin,, = 800 fmol/#g protein). Increasing the ionic strength of the medium, or phosphorylating the tail domain, decreased the affinity of this binding without altering the B ... amount of synapsin I rebound at saturation, representing ~6 % of the total synaptic vesicle protein, was similar to the amount found in native synaptic vesic...

Phosphorylation of Synapsin I by cAMP-Dependent Protein Kinase Controls Synaptic Vesicle Dynamics in Developing Neurons

“…Indeed, synapsin phosphorylation is regulated by synaptic activity (Nestler and Greengard, 1982;Sihra et al, 1989) and is associated with profound changes in its affinity for SVs and actin (Benfenati et al, 1989;Valtorta et al, 1992;. Consistently, phosphorylated synapsin I dissociates from SVs and diffuses in the axon during high-frequency stimulation (Torri Tarelli et al, 1992;Chi et al, 2001).…”

Synaptic activity slows vesicular replenishment at excitatory synapses of rat hippocampus

“…The synapsin family of phosphoproteins, which tethers vesicles to actin filaments, has been studied the most (Benfenati et al 1989;Greengard et al 1993;Pieribone et al 1995). It is generally thought that in response to Ca ??…”

Synaptic Ca2+in Darkness Is Lower in Rods than Cones, Causing Slower Tonic Release of Vesicles

“…This similarity also suggests that few, if any, vesicles failed to accumulate dye during the initial loading of gecko rods. Indeed, neither rods nor cones contain synapsin (Mandell et al, 1990), the key protein that immobilizes vesicles and therefore holds them in reserve at conventional synapses (Huttner et al, 1983;Bähler and Greengard, 1987;Benfenati et al, 1989), suggesting the lack of a reserve pool in photoreceptors.…”