Article Figures & Data
Figures
- Fig. 1.
Alignment of SVOP and SV2 sequences. The amino acid sequences of rat (R), C. elegans (C), andDrosophila (D)SVOPs, rat SV2A, SV2B, andSV2C, and an organic cation transporter (OCT1) from rat are aligned with each other. Gaps are indicated by hyphens. Residues conserved in >50% of all sequences are shown on a red background, residues conserved only in SVOPs are on a green background, and residues conserved in SV2 isoforms are on a bluebackground. The 12 transmembrane domains are marked by numbered lines above the sequences. Asparagine residues in N-linked glycosylation consensus sequences are shown in black. Sequences are named on theleft and numbered on the right. TheC. elegans and Drosophila SVOP sequences were assembled from genomic databanks. The N-terminal exon of theDrosophila SVOP sequence is absent because it could not be identified in the genomic sequence. The SV2A and SV2B sequences are from Bajjalieh et al. (1993), and the SV2C sequence is from Janz and Südhof (unpublished observations).
- Fig. 2.
Phylogenetic tree of the SVOP/SV2 gene family: relation to transport proteins. Genes are identified on theright by their GenBank accession numbers. The substrates for known transporters and the species of origin for each gene are listed in parentheses on the right. Genes were grouped into classes based on their nearest-neighbor relations. Note that SV2, SVOP, the organic cation and anion transporters, and a single bacterial sequence, the YceI gene from B. subtilis, form a single subgroup of related sequences. Bifurcation points were confirmed by bootstrapping; thenumbers of replications per 100 runs are shownnext to the bifurcation points. Names of sequences used in Figure 1 are bolded.
- Fig. 3.
Tissue distribution of SVOP expression. A rat multitissue RNA blot was hybridized with a cDNA probe for SVOP at high stringency and exposed for 4 hr (top) or 12 hr (bottom). Two mRNA species are observed only in brain even after long exposures (filled arrowheads). The single cross-hybridizing mRNA observed in testis (open arrow) is probably an artifact because it does not correspond in size to SVOP brain mRNAs and because a similar band is also observed with other unrelated probes in testis.
- Fig. 4.
Expression of SVOP in transfected COS cells and brain: specificity of antibodies. COS cells transfected with SV2A (lane 1), SV2B (lane 2), SV2C (lane 3), SVOP (lane 4), and control expression vectors (lane 5) and rat brain homogenate (lane 6) were analyzed by SDS-PAGE and immunoblotting using a polyclonal antibody against SVOP (top) or the monoclonal antibody against SV2 (bottom). Positions of specific bands are indicated byarrowheads on the right. The cross-reacting unrelated band observed with the SVOP antibody in COS cells but not in brain is marked by an open arrow.Numbers on the left indicate positions of molecular weight markers. Note the multiple heterogeneous bands for SV2A, SV2B, and SV2C in transfected COS cells that are caused by incomplete glycosylation (Janz and Südhof, unpublished observations).
- Fig. 5.
SV2 but not SVOP is N-glycosylated. Rat brain proteins (20 μg/lane) were incubated with (left lanesfor SVOP and SV2) or without (right lanes for SVOP and SV2) PNGase F. Protein extracts were analyzed by SDS-PAGE and immunoblotting for SVOP and SV2 as indicated. Molecular weight standards are indicated on the right.
- Fig. 6.
Developmental time course of SVOP and SV2 expression in brain. Equivalent amounts of rat brain protein from embryonic day 19 (E19), postnatal days 1, 4, 11, and 18 (P1, P4, P11, andP18), and adult animals were analyzed by immunoblotting with the polyclonal SVOP (top) and the monoclonal SV2 (bottom) antibody. Numbers on theleft indicate positions of molecular weight markers.
- Fig. 7.
Analysis of the localization of SVOP by subcellular fractionation. The following rat brain fractions were analyzed: brain homogenate (Total); low-speed pellet (P1); crude synaptosomes (P2); supernatant of synaptosomal fraction (S2); low-speed pellet of lysed synaptosomes containing synaptic plasma membranes, myelin, and mitochondria (LP1); high-speed pellet from lysed synaptosomes enriched in synaptic vesicles (LP2); synaptosomal cytosol (LS2); and synaptic vesicles purified from LP2 by controlled pore-glass chromatography (SV). Equivalent amounts of each fraction were immunoblotted with antibodies to SVOP (top) and SV2 (middle). The SV2 blot was reprobed with antibodies to NMDA-receptor and munc18–1 (bottom) to show that these proteins de-enrich during synaptic vesicle purification. Numbers on theright indicate positions of molecular weight markers.
- Fig. 8.
Copurification of SVOP with synaptogyrin on immunoprecipitated synaptic vesicles. Synaptic vesicles were captured on immunobeads from brain homogenates (Total) with beads coated with glycine only (Glycine-Beads; control) or with antibodies to synaptotagmin (Synaptotagmin-Beads) or synaptobrevin (Synaptobrevin-Beads). Bead fractions were immunoblotted for SVOP (top) or synaptogyrin (bottom).
- Fig. 9.
Immunocytochemical localization of SVOP in rat cerebellum. Sagittal sections were stained with the following antibodies using peroxidase detection with heavy metal enhancement: polyclonal SVOP antibody without additions (A), SVOP antibody incubated with MBP-SVOP fusion protein as a specific blocking agent (B), SVOP antibody incubated with MBP-cellugyrin fusion protein as a nonspecific blocking agent (C), or SV2 monoclonal antibody (D). Note the exact correspondence in staining patterns between SVOP and SV2. Scale bar, 0.5 mm.
- Fig. 10.
Immunocytochemical localization of SVOP in hippocampus and cortex. Rat brain sections were stained by immunoperoxidase labeling with polyclonal antibody to SVOP (A), SVOP antibody blocked with MBP-SVOP fusion protein (B), SVOP-antibody blocked with MBP-cellugyrin fusion protein (C), or SV2 monoclonal antibody (D). Scale bar, 0.5 mm.
- Fig. 11.
Comparison of the distributions of SVOP and SV2 in rat brain cortex. Sections were stained with polyclonal SVOP antibody (A, C) or monoclonal SV2 antibody (B, D). Cortical layers are identified in A and B. Cand D show a higher magnification of layer 5 fromA and B. The large pyramidal cells stained with the SVOP antibody but not the SV2 antibody are marked witharrows (C, D). Scale bars: A, B, 0.25 mm;C, D, 25 μm.
- Fig. 12.
Relative distributions of SVOP and SV2 in adrenal chromaffin granules and microsomes containing synaptic-like microvesicles. Equivalent amounts of protein from bovine brain (lane 1), bovine chromaffin granules (lane 2), and bovine adrenal medulla microsomes (lane 3) were analyzed by immunoblotting with antibodies to the indicated proteins. Numbers on the leftindicate positions of molecular weight markers.
