Synaptobrevin 2 Is Palmitoylated in Synaptic
Vesicles Prepared from Adult, But Not from
Embryonic Brain
Michael Veit,*
,1,2
Anja Becher,
†,1
and Gudrun Ahnert-Hilger
†
*Department of Immunology and Molecular Biology, Free University, Berlin; and
†
Department of Anatomy, Charité, Humboldt-University, Berlin, Germany
Neuronal SNARE-proteins such as synaptobrevin, SNAP
25, and synaptotagmin are key players during neurose-
cretion. So far palmitoylation of SNAP-25 and synaptotag-
min 1 have been described in vivo. Here we have analyzed
palmitoylation of the SNARE-proteins synaptobrevin 2
and synaptotagmin in vitro using synaptosomal and syn-
aptic vesicle preparations from rat brain. Labeling of syn-
aptic vesicles prepared from adult brain with [
3
H]palmi-
tate revealed synaptobrevin 2 besides synaptotagmin 1 as
major palmitoylated proteins. [
3
H]Palmitoylation of synap-
tobrevin 2 was resistant to chloroform/methanol extrac-
tion, but sensitive to reducing agents indicating a covalent
fatty acid bond to cysteine residues. Palmitoylation of
synaptobrevin 2 was also confirmed using endogenous
synaptobrevin 2 present in PC-12 cells and synaptobrevin
2 expressed with a vacciniavirus system in Cos cells. In
contrast to the situation seen with membrane prepara-
tions obtained from adult brain, synaptic vesicles pre-
pared from embryonic rat brain did not support [
3
H]palmi-
toylation of synaptobrevin and synaptotagmin. These
results suggest, that both synaptobrevin 2 and synapto-
tagmin were efficiently palmitoylated from mature synap-
tic vesicles. However, at least one component of the pal-
mitoylation machinery is developmentally upregulated.
INTRODUCTION
Neuronal SNARE-proteins have been identified as
key players in neurosecretion. They are located on syn-
aptic vesicles (v-SNARE: synaptobrevin 2, also called
VAMP 2) or on the presynaptic plasma membrane (t-
SNAREs: syntaxin 1 and SNAP-25). Intimate protein–
protein interactions between the SNARE-proteins
might be responsible for the attachment and fusion of
vesicles with the plasma membrane (Hanson et al., 1997;
Söllner et al., 1993; Sutton et al., 1998; Weber et al., 1998).
Another essential protein located on synaptic vesicles is
synaptotagmin 1, the likely calcium-sensor for neuro-
transmission. Fusion is probably triggered by an inter-
action of the SNARE-proteins with synaptotagmin.
Based on its ability to bind cytosolic SNAP-proteins,
synaptotagmin may be considered a specialized
SNARE-protein (SNAP-receptor) (Schiavo et al., 1995;
Südhof and Rizo, 1996).
The regulation and fine-tuning of these protein–pro-
tein interactions are now beginning to be elucidated.
Besides being a component of the SNARE complex,
synaptobrevin also binds to synaptophysin and its
closely related isoform synaptoporin, major residents of
synaptic vesicle membrane (Edelmann et al., 1995). The
presence of synaptobrevin in the two complexes is mu-
tually exclusive. However, the synaptophysin–synapto-
brevin interaction is not detectable in embryonic brain
showing that a modulation of SNARE-complex forma-
tion by synaptophysin is upregulated later during de-
velopment (Becher et al., 1999).
Lipid modification of SNARE-proteins might also con-
tribute to the regulation of the synaptic vesicle cycle. Syn-
aptotagmin and SNAP-25 are palmitoylated at cysteine
residues in vivo (Chapman et al., 1996; Hess et al., 1992; Veit
et al., 1996). Furthermore, palmitoylation of the yeast syn-
aptobrevin-homologues snc-1 and snc-2 has also been de-
scribed, but possible palmitoylation of synaptobrevin has
not been analyzed so far. Yeast and neuronal v-SNAREs
differ in the location of their cysteine residues, the poten-
tial site for palmitoylation (Couve et al., 1995).
The enzymology of protein-palmitoylation is poorly
understood. Two putative palmitoyl-transferases have
1
Both authors contributed equally to the paper.
2
To whom correspondence and reprint requests should be ad-
dressed. Fax: 49-30-2093 6171. E-mail: mveit@city.vetmed.fu-
berlin.de.
doi:10.1006/mcne.1999.0830, available online at http://www.idealibrary.com on
Molecular and Cellular Neuroscience 15, 408–416 (2000)
MCN
1044-7431/00 $35.00
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