Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

Abstract

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-offunction mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-toarginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/ VAChT and synaptobrevin. C OORDINATED muscle contraction in both vertebrates and nematodes requires release of the neurotransmitter acetylcholine (ACh) from motor neurons. ACh is synthesized in motor neurons by the enzyme choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular ACh transporter (VAChT). Following transmitter release, cholinergic signaling is terminated primarily through the action of acetylcholinesterase (AChE), which hydrolyses the released ACh into choline and acetyl-CoA. In the nematode Caenorhabditis elegans, the ChAT and VAChT proteins are encoded by the cha-1 and unc-17 genes, respectively (Alfonso et al. , 1994b. Mutations that functionally eliminate either protein are lethal , while reduction-of-function mutations result in animals that are small, slow growing, uncoordinated, and resistant to AChE inhibitors such as aldicarb .The unc-17 missense mutation e245 replaces a glycine with an arginine (G347R) in the ninth transmembrane domain (TMD9) of the VAChT protein . Genetic screens designed to identify mutations that improve the locomotion of e245 homozygotes have identified several loci that suppress the deleterious effects of the VAChT G347R mutation. Sandoval et al. (2006) found that the sup-8 locus corresponds to the previously characterized snb-1 gene, which encodes the v-SNARE synaptobrevin. The single sup-8 allele is associated with a missense mutation that results in a charge alteration (I97D) in the TMD of synaptobrevin. T...

Local microtubule organization promotes cargo transport in C. elegans dendrites

“…(3) that is be relatively AT rich to mimic intronic sequence; and (4) that it should not contain splice acceptor sites in the reverse orientation. Therefore, we used the 103-amino-acid sup-1(e995) mutant gene that can dominantly rescue the strongly uncoordinated phenotype of unc-17(e245) (Mathews et al, 2012). We designed a sup-1 minigene that can be inserted in introns as a geneediting marker (Fig.…”

Spontaneous Vesicle Fusion Is Differentially Regulated at Cholinergic and GABAergic Synapses

“…At each [Ca 2+ ] e level, the mEPSCs and mIPSCs were comparable in frequency and amplitude, suggesting a balance in synaptic activity between cholinergic and GABAergic synapses (Figures 1C and 1D; Table S1). This synaptic balance determines the behavior of the animals because disrupting synaptic transmission at either cholinergic or GABAergic synapses results in a strong uncoordinated behavioral phenotype (Mathews et al, 2012). However, whether the spontaneous vesicle fusion that occurs at these two types of synapses is regulated by similar or different molecular mechanisms has rarely been clarified.…”

Drosophila melanogaster as a genetic model system to study neurotransmitter transporters

“…In C. elegans , mutations in VAChT/unc-17 have already been used to screen for interacting loci (Mathews et al, 2013; Sandoval et al, 2006). One screen identified a mutant allele of synaptobrevin and a second screen identified the novel gene sup-1 (Mathews et al, 2013; Sandoval et al, 2006).…”

“…One screen identified a mutant allele of synaptobrevin and a second screen identified the novel gene sup-1 (Mathews et al, 2013; Sandoval et al, 2006). The nature of these second site mutant alleles suggest that in both cases the interactions are likely to be mediated by direct protein-protein binding to VAChT.…”

“…Another advantage is that it is not strictly hypothesis based. The authors of the VAChT interaction studies did not know that they would detect synaptobrevin, much less sup-1 (Mathews et al, 2013; Sandoval et al, 2006). Similarly, we suggest that identification of molecules that indirectly influence the function of neurotransmitter transporters in ways that we are unable to predict will be a very important future use for Drosophila .…”