Synaptic vesicles (SVs) store neurotransmitters and release them by exocytosis. The vesicular neurotransmitter transporters discriminate which transmitter will be sequestered and stored by the vesicles. However, it is unclear whether the neurotransmitter phenotype of SVs is solely defined by the transporters or whether it is associated with additional proteins. Here we have compared the protein composition of SVs enriched in vesicular glutamate (VGLUT-1) and GABA transporters (VGAT), respectively, using quantitative proteomics. Of >450 quantified proteins, ∼50 were differentially distributed between the populations, with only few of them being specific for SVs. Of these, the most striking differences were observed for the zinc transporter ZnT3 and the vesicle proteins SV2B and SV31 that are associated preferentially with VGLUT-1 vesicles, and for SV2C that is associated mainly with VGAT vesicles. Several additional proteins displayed a preference for VGLUT-1 vesicles including, surprisingly, synaptophysin, synaptotagmins, and syntaxin 1a. Moreover, MAL2, a membrane protein of unknown function distantly related to synaptophysins and SCAMPs, cofractionated with VGLUT-1 vesicles. Both subcellular fractionation and immunolocalization at the light and electron microscopic level revealed that MAL2 is a bona-fide membrane constituent of SVs that is preferentially associated with VGLUT-1-containing nerve terminals. We conclude that SVs specific for different neurotransmitters share the majority of their protein constituents, with only few vesicle proteins showing preferences that, however, are nonexclusive, thus confirming that the vesicular transporters are the only components essential for defining the neurotransmitter phenotype of a SV.

The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoformStx1Band RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose–response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similarK_d(dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho‐inactivating C3 transferase (C3bot) from Clostridium botulinum . Recently, we reported that transferasedeficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154‐182) from the C‐terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at sub‐micromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid‐ to higher‐order segments. C3bot ^154‐182 also increased the number of synaptophysin‐expressing terminals, up‐regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)‐transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot ^154‐182 on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full‐length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal‐entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C‐terminal peptide fragment covering aa 154‐182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full‐length C3, this C3 peptide selectively acts on neurons but not on glial cells. Holtje, M., Djalali, S., Hofmann, F., Munster‐Wandowski, A., Hendrix, S., Boato, F., Dreger, S. C., Große, G., Henneberger, C., Grantyn, R., Just, I., Ahnert‐Hilger, G. A 29‐amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation. FASEB J . 23, 1115–1126 (2009)

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked α‐(1→2) to galactose, β‐(1→3)‐glucans and arabinogalactan‐proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan‐proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.