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Comparative Study
. 2000 Feb 1;97(3):1101-6.
doi: 10.1073/pnas.97.3.1101.

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

P A Skehel  1 , R Fabian-Fine, E R Kandel
Affiliations
Comparative Study

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

P A Skehel et al. Proc Natl Acad Sci U S A. .

Abstract

VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum-resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis.

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Figures

Figure 1
Figure 1
Comparison of deduced VAP33 protein sequences from mouse, human, rat, and Aplysia. The regions of the major sperm protein homology and the predicted coiled-coil domains are indicated by thin and thick underscores, respectively. Identical and similar residues are shaded. Asterisks mark the positions of two conserved glycines that may function in coiled-coil interactions between transmembrane domains. mVAP33 GenBank accession no. AF157497.
Figure 2
Figure 2
Tissue distribution of mVAP33 mRNA expression. Total RNA from the tissues indicated was analyzed by Northern blot hybridization. Parallel blots were hybridized with radiolabeled mVAP33 or mouse β-actin cDNAs. mVAP33 transcripts of ≈2 kilobases are detectable in all tissues analyzed.
Figure 3
Figure 3
(A) Western blot analysis of fractionated mouse brain homogenate with mVAP33-specific polyclonal antiserum. (i) The major immunoreactive signal of ≈33 kDa is enriched in the pelleted fractions, but it is only partially extracted into the soluble fraction by 1% (vol/vol) Triton X-100. The cofractionating 19-kDa signal may be the result of proteolysis. (ii) In contrast, the t-SNARE syntaxin, although present in P1 and P2, is almost completely extracted by Triton X-100. The lower-molecular-weight signal is the result of incomplete stripping of the mVAP33 antisera. Molecular markers are Kaleidoscope Prestained Standards (Bio-Rad). (B) The 33-kDa immunoreactivity is specific for mVAP33. The antisera were preincubated with Ni-NTA-agarose with or without recombinant protein. Ni-NTA-agarose, or Ni-NTA-agarose and His6-tagged recombinant green fluorescent protein (EGFP) have no affect, whereas His6-tagged recombinant mVAP33 specifically depletes the 33-kDa signal. (C) mVAP33 is an integral membrane protein. P2 was extracted with 1.5% Triton X-114 and separated into aqueous (A) and detergent (D) phases at 37°C. The 33-kDa immunoreactivity partitions into the detergent phase, indicating that, like the molluscan protein, mVAP33 is an integral membrane protein. After Triton X-114 extraction, mVAP33 appears to form an SDS-stable oligomer of approximately 66 kDa.
Figure 4
Figure 4
Indirect immunofluorescence analysis of cultured cortical cells. Cortical cells were prepared from P2 Wistar rats and maintained in culture for 7 days before fixation and analysis. Cultures were stained with the specific antisera indicated. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic terminals, but has a reticular dstribution in glia and neurons. There is extensive colocalization of mVAP33 and the ER protein calnexin. At higher power, mVAP33 immunoreactivity shows a punctate distribution distinct from that of calnexin.
Figure 5
Figure 5
Immunogold labeling of hippocampal organotypic cultures for mVAP33. Immunoreactivity was mainly associated with microtubules (A), where it was often clustered (B and C). Clustered labeling was also associated with the membranes of vesicular structures (D and E). Gold particles were also occasionally seen between a vesicle and a microtubule (F). There was no detectable signal associated with synaptic vesicles. (Bars: A, 300 nm; B and D, 200 nm; C, 100 nm; E, 150 nm; and F, 80 nm.)
Figure 6
Figure 6
Immunogold labeling of mVAP33 in cultured cortical cells. Neuronal processes contained large numbers of heterogeneously sized membrane-bound structures (A, B, and C, arrows) that were frequently labeled. Labeling often coincided at electron-dense structures (D, arrow). Larger extended aggregates of mVAP33 immunoreactivity were occasionally detected in short filamentous structures that were frequently localized with membrane structures (E and F, arrows). (Bars: A and C, 400 nm; B, 650 nm; D, 250 nm; E, 850 nm, and F, 550 nm.)
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