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Comparative Study
. 2010 Jan 6;30(1):2-12.
doi: 10.1523/JNEUROSCI.4074-09.2010.

Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein

Mads Grønborg  1 , Nathan J Pavlos, Irene Brunk, John J E Chua, Agnieszka Münster-Wandowski, Dietmar Riedel, Gudrun Ahnert-Hilger, Henning Urlaub, Reinhard Jahn
Affiliations
Comparative Study

Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein

Mads Grønborg et al. J Neurosci. .

Abstract

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, approximately 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.

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Figures

Figure 1.
Figure 1.
Immunoisolation of SVs using VGLUT-1- and VGAT-specific antibodies. Immunoblot analysis shows that VGLUT-1-isolated vesicles are largely depleted for VGAT vesicles (top panel) and VGAT-isolated vesicles are largely depleted of VGLUT-1 vesicles (bottom panel). LP2, Enriched synaptic vesicle fraction; IP, immunoprecipitated sample; FT, flow through (supernatant) after IP.
Figure 2.
Figure 2.
Quantitative proteomic comparison of immunoisolated VGLUT-1- and VGAT-specific SVs reveals only few differences. Immunoisolated fractions were digested by trypsin and labeled with iTRAQ 114 or 115, respectively. The labeled peptides were first fractionated by SCX and subsequently analyzed by reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). A–C, A total of 460 proteins were quantified in our study (supplemental Table 1, available at www.jneurosci.org as supplemental material), including vesicular transporters and ion channels (A) and trafficking and SV membrane proteins (B, C). Dotted lines indicate a ratio of 1, i.e., no difference between the two vesicle populations. Proteins identified to be significantly differentially expressed include in addition to the transporters VGLUT-1 and VGAT, the zinc transporter ZnT3, the vesicle membrane proteins SV2B, SV2C, SV31, synaptophysin, several synaptotagmin isoforms, the SNARE syntaxin 1a, and MAL2, a novel SV protein.
Figure 3.
Figure 3.
Differential enrichment of selected proteins on VGLUT-1- and VGAT-specific SVs, respectively, monitored by immunoblotting. Synaptotagmin 1, MAL2, and SV2B are enriched on VGLUT-1 vesicles, whereas synapsin 1 and SV2C are enriched in VGAT vesicles. VGLUT-2, syb 2 (VAMP2), SV2A, and Rab 3a all showed equal protein distribution in the two fractions. These data corroborate the findings obtained by quantitative MS (Fig. 2).
Figure 4.
Figure 4.
Association of differentially distributed SV proteins with glutamatergic and GABAergic nerve terminals in primary cultures of hippocampal neurons. Twelve DIV hippocampal neurons were double labeled with either VGLUT-1 or VGAT in combination with synaptophysin (syp), synapsin, SV2A, SV2B, and SV2C. Top panel, Double-labeling with VGLUT-1 and VGAT reveals non-overlapping punctuate staining showing that these transporters do not occur in the same nerve terminal. Synaptophysin (Syp) and Synapsin I, both ubiquitously expressed SV proteins, show a high degree of colocalization with both VGLUT-1 and VGAT. As expected, a similar result was obtained for SV2A. In contrast, SV2B colocalizes with VGLUT-1 but not with VGAT. SV2C, however, again colocalizes well with both VGLUT and VGAT.
Figure 5.
Figure 5.
Colocalization of SV2 isoforms with VGLUT-1 and VGAT in sections of mouse cerebellum. A–F, Fluorescence microscopic analysis of SV2A (A, B), SV2B (C, D), and SV2C (E, F) all stained in red and either VGLUT-1 (A, C, E) or VGAT (B, D, F) stained in green. Scale bar, 100 μm.
Figure 6.
Figure 6.
Biochemical characterization of MAL2. A, MAL2 enriches preferentially in the detergent phase during TX-114 phase separation indicating that it contains detergent-binding (transmembrane) domains. Rat brain homogenate (100 μg) was dissolved in 10% precondensated TX-114, and after phase separation the phases were analyzed by SDS-PAGE and immunoblotting using a MAL2-specific polyclonal antibody. B, The N terminus of MAL2 is exposed on the vesicle surface. An enriched SV fraction (LP2) was either left untreated (left panel) or digested with Pronase (an unspecific protease) (right panel). Untreated MAL2 gives a distinct band ∼20 kDa as expected, whereas the epitope (indicated by a green box in the right drawing) is not detectable after Pronase treatment. To ensure that luminal domains remained protected during protease treatment, we also probed for proteolysis of synaptotagmin I using monoclonal antibodies specific either for the cytoplasmic (orange) or luminal (red) regions. As expected, fragments carrying the N-terminal epitope persisted whereas the C-terminal epitope was destroyed, confirming the selectivity of the proteolysis. C, MAL2 copurifies with SV proteins during subcellular fractionation. MAL2 cofractionates with bona-fide vesicle proteins such as synaptophysin, Rab3a, VGLUT-2, and Syt1. Note that the final fraction is devoid of contamination by postsynaptic membranes (NMDA-R1) whereas Mint, a soluble protein, is detectable but does not show enrichment. H, Homogenate; P1, crude nuclear pellet; S2, soluble fraction; P2′, crude synaptosomes; LP1, synaptosomal membrane fraction; LP2, crude SVs; PK1 (peak 1) and CPG, larger membranes and pure SVs, respectively, after separation by size exclusion chromatography on Controlled-Pore Glass beads (CPG). D, Immunogold electron microscopy analysis of SVs (negative staining) reveals colocalization of both MAL2 (10 nm gold) and VGLUT-1 (5 nm gold) on the same vesicles. E, A survey of MAL2 expression by immunoblotting in different rat tissues reveals that MAL2 is highly expressed in the brain cortex and liver. Moderate to low expression is observed in brain stem and cerebellum and low expression is observed for the rest of the tested tissues. Membrane proteins in the homogenate samples (corresponding to 100 μg of starting material) were extracted by TX-114 partitioning and analyzed by immunoblotting. The quantitative MAL2 immunoblot analysis was done in triplicates where the mean (±SD) is presented in the histograms. The MAL2 signal (highest signal) in brain cortex was set to 100%.
Figure 7.
Figure 7.
Association of MAL2 with glutamatergic nerve terminals in primary cultures of hippocampal neurons (see legend to Fig. 4 for details). A, Immunostaining for MAL2 (red channel) is observed in the soma, axons and dendrites of both glutamatergic (VGLUT-1, green stain) and GABAergic (VGAT, green strain) neurons. Magnified views reveals punctuate staining for MAL2 as expected for a SV protein (A, B) with high degree of colocalization with VGLUT-1 in contrary to VGAT. C, Correlative line-scans of synaptic boutons demonstrate selectivity of MAL2 for VGLUT-1-positive nerve terminals.
Figure 8.
Figure 8.
Immunofluorescence and EM analysis of MAL2 in rat and mouse hippocampal and cerebellar sections. A, B, Fluorescence microscopic analysis of MAL2 (red channel) with either VGLUT-1 (A) or VGAT (B) (green channel) in mouse hippocampal sections. A detail of the CA3 area attached to the overview reveals the colocalization between VGLUT-1 and MAL2 but not between VGAT and MAL2. Scale bar, 100 μm. C, D, Fluorescence microscopic analysis of MAL2 stained in red and either VGLUT-1 (C) or VGAT (D) stained in green in rat cerebellar cortex. VGLUT-1 perfectly colocalizes with MAL2 in the molecular layer while no overlap is seen between MAL2 and VGAT. E, Double immunogold labeling of a mossy fiber terminal from the rat hippocampal CA3 area indicates the synaptic and vesicular coexistence of MAL2 (10 nm gold) and VGLUT-1 (5 nm gold). The magnified images (two right panels) represents areas from independent samples are therefore not marked in the overview (left panel). Scale bar, 200 nm.
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