Abstract
Cup-shaped lipoprotein structures called porosomes are the universal secretory portals at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release intravesicular contents. In neurons, porosomes measure ∼15 nm and are comprised of nearly 40 proteins, among them SNAREs, ion channels, the Gαo G-protein and several structural proteins. Earlier studies report the interaction of specific lipids and their influence on SNAREs, ion channels and G-protein function. Our own studies demonstrate the requirement of cholesterol for the maintenance of neuronal porosome integrity, and the influence of lipids on SNARE complex assembly. In this study, to further understand the role of lipids on porosome structure-function, the lipid composition of isolated neuronal porosome was determined using mass spectrometry. Using lipid-binding assays, the affinity of porosome-associated syntaxin-1A to various lipids was determined. Our mass spectrometry results demonstrate the presence of phosphatidylinositol phosphates (PIP's) and phosphatidic acid (PA) among other lipids, and the enriched presence of ceramide (Cer), lysophosphatidylinositol phosphates (LPIP) and diacylglycerol (DAG). Lipid binding assays demonstrate the binding of neuronal porosome to cardiolipin, and confirm its association with PIP's and PA. The ability of exogenous PA to alter protein–protein interaction and neurotransmitter release is further demonstrated from the study.
Highlights
In cells it is estimated that a staggering number of lipids, more than 10,000 different species are present [1]
For example, target membrane proteins SNAP-25 and syntaxin-1A called t-SNAREs present at the base of neuronal porosomes, and a synaptic vesicle-associated membrane protein or v-SNARE, are part of the conserved protein complex involved in membrane fusion and neurotransmission
Neurotransmission is dependent on the transient fusion of 40–50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the base of 15 nm neuronal porosomes present at the pre-synaptic membrane in nerve terminals
Summary
In cells it is estimated that a staggering number of lipids, more than 10,000 different species are present [1]. For example, target membrane proteins SNAP-25 and syntaxin-1A called t-SNAREs present at the base of neuronal porosomes, and a synaptic vesicle-associated membrane protein or v-SNARE, are part of the conserved protein complex involved in membrane fusion and neurotransmission. Recent studies involving the crystal structure of a lipid-G-protein-coupled receptor [18] demonstrates that the lysophospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone, by activating the G-protein-coupled receptor. These few examples clearly demonstrate the critical role of membrane lipids on various cellular functions. To further determine lipid interactions with specific porosome proteins, lipid-binding/lipid overlay assays were performed and known lipid-binding domains within the porosome complex for possible lipid-protein interactions was assessed in this study
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