Genetic interactions between UNC-17/VAChT and a novel transmembrane protein in Caenorhabditis elegans.
The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine 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.
Genetic Models of Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Eleanor A
EA
Mathews
Gregory P
GP
Mullen
Jonathan
J
Hodgkin
Janet S
JS
Duerr
James B
JB
Rand
D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D016153:Genes, Suppressor; D005816:Genetic Complementation Test / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009420:Nervous System / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013569:Synapses / Q000235:genetics; D050494:Vesicular Acetylcholine Transport Proteins / Q000235:genetics* / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; C581333:Sup-1 protein, C elegans; C082365:Unc-17 protein, C elegans; D050494:Vesicular Acetylcholine Transport Proteins