Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers.

Abstract

Abstract. Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/#g of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kj value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenasesensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue l-palmitoyl-2- [11-[4-[3-(trifluoromethyl)] undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule. The results suggest that synapsin I, which has an amphiphilic character and local regions of positive charge, interacts withartificial membranes containing acidic phospholipids, and this surface interaction, presumably of electrostatic nature, is followed by a penetration of the head domain into the hydrophobic core of the membrane. p ROTEIN phosphorylation represents a process of paramount importance in the nervous system and is involved in signal transduction and information processing (45). Synapsin I, a collective name for two closely related peptides (Ia and Ib) with apparent molecular weights of 86,000 and 80,000 on SDS-PAGE, is a major neuronspecific phospho...

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons.

“…Association of synapsin I with fuzzy filamentous materials, most probably cytoskeletons (F-actin, microtubule, neurofilament, etc.) as the beginning of synapsin I transport, was supported by biochemical experiments (3,4,6,8,9,19,38) and by an in vitro reconstruction experiment (22). In particular, the latter experiment showed that synapsin I had high affinity to the cytoskeletons and could crossbridge them in vitro.…”

“…This apparent discrepancy mayreflect the sequence of synapsin I expression and the synaptic vesicle formation. Since synaptic vesicles or its integral proteins work as a synapsin I receptor (4,8,9,30,31,34,38), synapsin I will concentrate around the vesicles soon after the vesicles are formed. Therefore, since p65 was detected sooner than synapsin I by protein blot, synapsin I colocalizes with p65 if p65 or the vesicles integrating p65 have affinity with synapsin I.…”

A Gain-of-Function Mutation in Synaptotagmin-1 Reveals a Critical Role of Ca2+-Dependent SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Binding in Synaptic Exocytosis

“…The trypsinized membranes were then centrifuged at 150,000 ϫ g for 1 h, and the cytosolic domain of synaptotagmin-1 was recovered in the supernatant and stored at Ϫ80°C. Phospholipid binding assays were performed using heavy liposomes with a synaptic phospholipid composition and phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ) as indicated previously (Deutsch and Kelly, 1981;Benfenati et al, 1989;Rhee et al, 2005;Li et al, 2006). After SDS-PAGE, synaptotagmin-1 associated with heavy liposome was quantified by immunoblotting using Cl41.1 and 125 I-labeled secondary antibody.…”

Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy

“…The axonal button membranes are comprised of complex mixtures of lipids, however the predominant lipid classes are the phosphatidylcholines (PC) and the phosphatidylserine (PS) lipids, depending on location [4]. PC lipids in particular are commonly found in the outer leaflet of the synaptic vesicle [5] and, although not studied extensively in DA interactions, as a subclass of lecithin it is an essential precursor to acetylcholine that regulates signaling in the brain. The main function of choline and its metabolites is to transduce membrane signals and limited data available suggest that an increase in PC supplementation can alleviate the symptoms of psychiatric disorders, such as Schizophrenia, bipolar disorder, and Alzheimer's and Parkinson's [6,7].…”