Vesicle membrane protein analysis

The SNAREs involved in the fusion of synaptic vesicles and of secretory granules in neuroendocrine cells, referred to as neuronal SNAREs, have been intensely studied and serve as a paradigm for all SNAREs. They include syntaxin 1A and SNAP-25 at the presynaptic membrane and synaptobrevin 2 (also referred to as VAMP 2) at the vesicle membrane. Their importance for synaptic neurotransmission is documented by the fact that the block in neurotransmitter release caused by botulinum and tetanus neurotoxins is due to proteolysis of the neuronal SNAREs (Schiavo et al. 2000). Genetic deletion of these SNAREs confirmed their essential role in the last steps of neurotransmitter release. Intriguingly, analysis of chromaffin cells from KO mice lacking synaptobrevin or SNAP-25 showed that these proteins can be at least partially substituted by SNAP-23 and cellubrevin, respectively (Sorensen et al. 2003 Borisovska et al. 2005), i.e., the corresponding SNAREs involved in constitutive exocytosis. 

In some experiments vesicle membranes treated with phospholipase A and control vesicles were separated from the ACh in the suspension medium by gelfiltration on a Sephadex G-50 column. Vesicles or enzyme-treated vesicles were identified by protein analysis and came out with the void volume. After acid boiling it was found by bioassay that the enzyme-treated vesicles contained less ACh than the controls. 

The control could be established if at any one time there was at the membrane more vesicles than those that were fusing. However in order to sustain the increase in secretion it would be necessary to transmit the signal back to the synthetic system and packaging process at the Golgi apparatus so that a new steady state system was obtained. This would produce the requisite number of vesicles containing the polysaccharides necessary to maintain the new rate of fusion and secretion. The turnover of the Golgi apparatus can be very fast in plant cells and times of 5-40 minutes have been calculated (36). In vitro experiments which involved isolation of membrane fractions from maize-root cells also showed that Ca was necessary for membrane fusion (37). Analysis of the membranes indicated that the Ca dependence involved membrane proteins and one of these was a Ca and Mg -dependent ATPase (38). 

A first approach to study the interaction of posttranslational modified Ras proteins with membranes was the analysis of binding and exchange of isoprenyl-ated peptides with and between lipid vesicles utilizing a fluorescent bimanyl label. Studies with K-Ras peptides revealed that a single isoprenyl group is sufficient for membrane association only if supported by carboxymethylation of the C-terminal cysteine [227,228]. 

Other evidence is reported in the paper of Canellas et al. (2002), in which a stimulation of the plasma membrane H+-ATPase activity took place, apparently associated with an ability to promote expression of this enzyme, as confirmed by western blot analysis. Using antibodies raised against H+- ATPase PMA2 isoform from Nicotianaplumbaginifolia Viv. (Morsomme et al., 1996), it was discovered that the amount of immunoreactive protein at the PM A locus (approximately 96 kD) increased almost threefold in the membrane vesicles isolated from maize roots treated with HA. 

---