Membrane proteins interaction zones

In neurons, the SNARE complex consists of three main proteins the v-SNARE synaptobrevin or VAMP (vesicle-associated membrane protein), and two t-SNAREs, syntaxin and SNAP-25 (synaptosomal associated protein of 25 kD). Synaptobrevins traverse the synaptic vesicle membrane in an asymmetric manner a few amino acids are found inside the vesicle, but most of the molecule lies outside the vesicle, within the cytoplasm. Synaptobrevin makes contact with another protein anchored to the plasma membrane of the presynaptic neuron, syntaxin, which is associated with SNAP-25. Via these interactions, the SNARE proteins play a role in the docking and fusion of synaptic vesicles to the active zone. 

Both universal staining procedures and specific detection techniques can be performed after (electro) transfer or (electro)blotting of proteins (also called Western blotting) from the gel matrix (which sometimes hinders protein analysis) to a nitrocellulose or polyvinylidenedifluoride membrane, to which they are bound and immobilized. On the membrane, protein molecules are faster and better accessible for the interactions with the applied specific antibodies. The antigen-antibody complexes are visualized by a second antibody (against the first antibody) with an attached enzyme label, which catalyzes the color reaction in the place of the protein zone. 

Fig. 6.2 Interactions between phospholipids, membrane proteins, and flavonoids. (a) In acidic and neutral conditions, flavonoids such as quercetin intercalate between phospholipids in the hydrophobic zone and initiate the formation of an ordered lipid phase. Flavonoids can also interact with membrane proteins, (b) In alkaline conditions, deprotonated flavonoids cover the polar head surface of phospholipids and interact with membrane proteins, (c) When phloretin replaces quercetin, the distance between the hydrocarbon chains is increased and lipids form a superordered lipid phase. Intercalation of phloretin between the polar heads of phospholipids induces micelle formation, (d) Outer and inner phospholipidic layers can be interdigitated when phospholipids are spaced by phloretin but not by quercetin (a and b). Modified from Tarahovsky et al. (2008).

Fig. 8. Effects of disruption of endophilin and amphiphysin interactions on clathrin-mediated endocytosis at the reticulospinal synapse. (A) Electron micrograph of the lateral side of the active zone in a control synapse stimulated at 5 Hz. Note the presence of clathrin-coated pits with different shapes. (B) Electron micrograph of the comparable area of a synapse in an axon that was stimulated at 5 Hz for 30 min after injection of endophilin antibodies. Note the pocket-like membrane expansions (arrows) at the margin of the synaptic area and the appearance of numerous shallow coated pits (arrows). (C) A synapse in an axon which was stimulated at 0.2 Hz for 30 min after injection of a fusion protein containing the SH3 domain of amphiphysin linked to GST. Note the accumulation of constricted coated pits around the active zone. Scale bar, 0.2 pm. B, modified from Ringstad et al. (1999), Neuron 24, 143-154, with permission copyright is held by Cell Press. C, modified from Shupliakov et al. (1997a) Science 276 259-263, with permission copyright 1997 AAAS.

Serotypes B, D, E, and G cleave different sites on the synaptic vesicle protein, synaptobrevin (VAMP), whereas serotypes A and E cleave the presynaptic membrane-associated protein SNAP-25 (Schiavo et al., 2000 Simpson, 2004). Serotype Cl is unique in that it cleaves two cytoplasmic proteins, syntaxin and SNAP-25 (Wiltiamson et al., 1996). Interaction of these SNAREs on the surface of synaptic vesicles and active zone membranes is required for voltage- and Ca " -dependent release of neurotransmitter cleavage by BoNT inhibits this process, leading to muscle weakness and paralysis (Sutton et al., 1998 Schiavo et al., 2000). Cleavage of SNARE proteins appears to be sufficient to account for all actions of the BoNTs, and the SNARE hypothesis has received near universal acceptance since its introduction in the early 1990s. 

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