Invertebrate Synapsins: A Single Gene Codes for Several Isoforms inDrosophila

Abstract

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

Homo- and Heterodimerization of Synapsins

“…The various synapsins are composed of similar N-terminal and central domains followed by variable C-terminal domains (3). Their most striking feature is the presence of a large central domain, the C-domain, which binds ATP with high affinity and is evolutionarily the most conserved domain of synapsins (3,10,16,17). Although the precise functions of synapsins are unknown, analysis of knockout mice revealed an essential role for synapsins I and II in regulating synaptic vesicle exocytosis and in stabilizing synaptic vesicles (13)(14)(15).…”

“…The largest domain of synapsins is the central C-domain, which measures more than 300 residues and is the only domain that is conserved in invertebrate synapsin (10). The C-domain is flanked on the N terminus by short A-and B-domains; the A-domain contains the only phosphorylation site that is present in all synapsins.…”

Stoned B mediates sorting of integral synaptic vesicle proteins

“…After permeabilization with 0.1% Triton X-100/PBS, preparations were incubated with primary antibodies at 4°C overnight. We used the following antibodies: anti-myc (1:500), anti-STNA/B (1:1000), anti-synaptotagmin1 (1:2000), anti-synaptobrevin (1:1000; Sweeney et al, 1995), anti-synapsin (1:200;Klagges et al, 1996; kind gift by Dr. E. Buchner), anti-vGLUT (1:500; kind gift by Dr. A. DiAntonio), anti-DAP160 (1:1000), and anti-horseradish peroxidase (HRP) (1:500; texas red conjugated; Jackson ImmunoResearch). Secondary anti-mouse and anti-rabbit antibodies (alexa488/633 conjugated) were obtained from Molecular Probes and were typically used at a dilution of 1:500 (2 hrs at room temperature).…”

Mas‐allatotropin in the developing antennal lobe of the sphinx moth Manduca sexta: Distribution, time course, developmental regulation, and colocalization with other neuropeptides

“…The result suggests a high specificity of the antiserum for all tested brain homogenates. The MW of 70 and 74 kDa for two isoforms of Synapsin 1 of Drosophila was shown by Klagges et al (1996) in Western blots of whole head homogenates. An additional band at 80 kDa as shown by Klagges et al (1996) was not detected, suggesting that this isoform is not highly enough expressed in larval brain tissue of Drosophila to be detected in Western blot.…”