Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability

Abstract

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

Figure 1

Eye phenotypes of syb and n-syb. To analyze eye phenotypes by scanning electron microscopy, somatic recombination with the EGUF/hid method was used to make the entire eye and only the eye homozygous for either a wild-type (A), syb¹⁴⁴ (B), or n-syb^ΔF33B (C) chromosome. (A) The control eye is slightly roughened and reduced in consequence of the recombination and cell death entailed in the EGUF/hid method. (B) syb¹⁴⁴ ablates all or nearly all the ommatidia, and the eye is reduced to a scar. (C) In contrast, n-syb^ΔF33B appears similar to control. (D Left) ERG of a control eye similar to that in A illustrating the response to a 1-s flash of light (bar). On and off transients corresponding to synaptically evoked responses are marked by arrows. (D Right) Similar ERG recording from an eye homozygous for n-syb^ΔF33B, as in C. A robust response to light is obtained. On and off transients, however, were always completely absent (arrows). Specific genotypes were y,w;FRT42D, GMR-hid/FRT42D;ey-GAL4,UAS-FLP/+ (A), y,w;FRT42D,GMR-hid/FRT42D, syb¹⁴⁴;ey-GAL4,UAS-FLP/+ (B) y,w;ey-GAL4,UAS-FLP/+;FRT80B,GMR-hid/FRT80B,n-syb^ΔF33B (C and D Right), and y,w;ey-GAL4,UAS-FLP/+;FRT80B, GMR-hid/FRT80B (D Left).

Figure 2

Both n-Syb and Syb form SDS-resistant complexes. Recombinant SNAP-25, syntaxin 1A, and either Syb or n-Syb were allowed to form complexes, and the temperature sensitivity of the complex in SDS was assayed. Complexes were visualized with antibodies to syntaxin. The position of monomeric syntaxin is marked, as are the positions of the SNARE complexes. A small amount of immunoreactivity at ≈55 kDa is due to uncleaved GST-syntaxin that comigrates with SNARE complex 1 but does not depend on the presence of other SNARE partners.

Figure 3

The cell lethality of a syb mutation in the eye was rescued by other synaptobrevins. (A) Scanning electron micrograph of a control eye with no syb mutation. (B) An eye that is homozygous for syb¹⁴⁴ lacks most ommatidia. syb mutant eyes, were rescued with transgenes bearing syb (C), n-syb (D), rat VAMP2 (E), and rat cellubrevin (F). Specific genotypes were y,w;FRT42D,GMR-hid/FRT42D;ey-GAL4,UAS-FLP/+ (A), y,w;FRT42D,GMR-hid/FRT42D,syb¹⁴⁴;ey-GAL4,UAS-FLP/+ (B), y,w;FRT42D,GMR-hid/pHs-sybB-2-1,FRT42D,syb^21-15; ey-GAL4,UAS-FLP/+ (C), y,w;FRT42D,GMR-hid/pHs-n-sybII-10-2,FRT42D,syb^21-15;ey-GAL4,UAS-FLP/+ (D), y,w;FRT42D, GMR-hid/pUAST-VAMP2,FRT42D,syb^21-15;ey-GAL4,UAS- FLP/+ (E), and y,w;FRT42D,GMR-hid/pUAST-cellubrevin FRT42D,syb¹⁴⁴;ey-GAL4,UAS-FLP/+ (F).

Figure 4

syb can rescue the ERG defect of n-syb. ERG recordings of n-syb⁺ (A) and n-syb homozygous null (B) eyes formed by somatic recombination by the EGUF/hid system. The n-syb mutation removes the on and off transients. However, the expression of a syb transgene restores the transient deflections as shown by the arrows in C. (A) y,w;ey-GAL4,UAS-FLP/+;FRT80B,GMR-hid/FRT80B; (B) y,w;ey-GAL4,UAS-FLP/+;FRT80B,GMR-hid/FRT80Bn-syb^ΔF33Bb; (C) y,w;ey-GAL4,UAS-FLP/pHs-sybB-2-1;FRT80B,GMR-hid/FRT80Bn-syb^ΔF33Bb.

Figure 5

syb and n-syb transgenes can restore synaptic transmission in n-syb null mutants. (A) Representative whole-cell recordings of nerve-evoked excitatory junctional currents in a wild-type embryo. (B) No evoked responses are observed in an n-syb mutant, but responses are restored partially by the presence of either n-Syb (C) or Syb (D) transgenes. In each panel, responses to 10 consecutive stimuli are shown. Specific genotypes are OrR (A), y,w;n- syb^ΔF33B (B), y,w;pHs-n-sybII-7-1;n-syb^ΔF33B (C), and y,w;pHs-sybB-2-1; n-syb^ΔF33B (D).