Author Summary The human genome project has given us a catalog of the genes that make a human; however, the function of about 40% of these genes remains elusive. Many of these mysterious genes have relatives in simpler organisms like worms and flies, where their function can be studied much more easily than in a mammal. Here, we investigate one such family of genes, called macoilins, using the worm C. elegans. We show that worm macoilin, like mouse macoilin, is expressed widely but specifically in nerve cells. We create worms in which the macoilin gene is defective and show that, although they retain a nervous system that looks normal, they have behavioral defects. We show that these behavioral defects reflect an inability of nerves to signal efficiently. Nerve signalling relies on calcium channels and the defect of macoilin mutants resembles that of animals defective in a particular calcium channel component. We find that in nerve cells the macoilin protein resides specifically in the ‘‘factory’’ that assembles nerve signalling molecules, including calcium channels. Our results suggest that macoilin either directly helps assemble an ion channel or is needed to make a channel regulator. Our work in worms provides a blueprint to investigate the function of macoilins in mammals. animal phyla, and highlighted their shared structural features. However the biological function of many of these conserved gene families remains mysterious. Genetic studies in model organisms such as Drosophila and C. elegans have provided a powerful way to functionally characterize novel conserved genes. This is exempli- fied by discovery of ion channel families, e.g. TRP [9], axon guidance pathways (UNC-6/netrin; UNC-40/DCC; ROBO [10]) and molecules involved in synaptic release (e.g. UNC-13 [11] and UNC-18 [12]). In all these cases genetic studies in flies or worms were recapitulated in mammals and catalyzed subsequent vertebrate work. Here, we functionally characterize, for the first time, a member of a conserved family of proteins called macoilins. We find a macoilin gene in all available genome sequences of animals, from placozoa to man, but not in yeast or Dictyostelium. C. elegans macoilin, like mouse macoilin [13], is expressed throughout the nervous system. In C. elegans expression begins embryonically at the time neurons are born, and persists to adulthood. Using antibodies and compartment specific markers we show that C. elegans macoilin is localized to the rough endoplasmic reticulum and is excluded from neurites. We identify multiple C. elegans mutants of macoilin and show that these have altered behavior, but normal development of the nervous system. Using Ca2+ imaging, we show that macoilin is required for cell-intrinsic neuronal excitability in the O2-sensing neuron PQR. This phenotype is mirrored in animals defective in EGL-19, the sole C. elegans L-type voltage gated ion channel (L-VGCC) alpha1 subunit. Our data suggest that macoilin is involved in assembly or traffic of ion channels or ion channel regulators. Results Mutations in C. elegans macoilin disrupt aggregation and are associated with multiple behavioral defects N2, the laboratory wild-type strain of C. elegans, feeds in isolation; however most wild-collected strains of this species feed in groups [14–15]. N2 animals fail to aggregate because of a gain-of- Macoilin: an ER Protein Regulating Neuron Activity function mutation in the neuropeptide receptor npr-1: if this receptor is knocked out they aggregate strongly [14][16]. To define genes that promote aggregation we mutagenized npr-1(null) animals and sought non-aggregating mutants. One complemen- tation group we identified comprised three recessive alleles, db1, db9 and db129, and defined a gene we called maco-1 (for macoilin-1 see below). All three maco-1 mutations strongly suppressed aggregation and bordering behaviors (Figure 1A) and disrupted the ability of npr-1 animals to switch between roaming widely and dwelling locally according to ambient O2 levels (Figure 1B) [17]. maco-1; npr-1 mutants were healthy and displayed strong attraction to diacetyl and benzaldehyde (Figure S1A, S1B), odorants that are detected by the AWA and AWC olfactory neurons respectively [18]. They also strongly avoided high osmotic potential, a response mediated by ASH nociceptive neurons (Figure S1C) [19]. Like N2 animals, maco-1 single mutants did not aggregate and only showed weak bordering behavior (Figure S1D). Closer examination, however, revealed additional behavioral phenotypes associated with maco-1 mutations. We quantified these phenotypes in the presence of the N2 allele of npr-1, since this is the standard genetic background. First, harsh touch to the head elicited significantly longer reversals in maco-1 mutants compared to N2 controls (Figure 1C). Second, maco-1 mutants exhibited swimming defects [20] characterized by a decrease in the frequency of body bends (Figure 1D), an increase in the amplitude of body bends (data not shown), and coiling (Figure 1E). maco-1 mutants also showed increased coiling when on an agar substrate (data not shown). Interestingly, the maco-1 locomotory defects increased with age (Figure S1E, S1F). Third, whereas N2 animals suppress egg-laying in the absence of food [21], this inhibition was partly relieved in db9 and db129 mutant animals (Figure 1F). These subtle but pleiotropic defects of maco-1 mutants suggest deficits in multiple neural circuits. We mapped maco-1 to a 30 kb interval on Chromosome I, close to D2092.5, a previously uncharacterized gene. DNA sequencing revealed that all three maco-1 alleles disrupted D2092.5 (Figure 2A). The db1 allele modified the splice donor site of intron 9 (G6024A); db9 changed the arginine codon at position 534 to a stop codon and is predicted to truncate the MACO-1 protein (Figure 2D); the db129 allele was associated with a mutation in the splice acceptor site of intron 2 (G595A). Together, these data suggest that maco-1 corresponds to D2092.5. This was confirmed by transgenic rescue of the maco-1 mutant phenotypes with a wild-type D2092.5 transgene (Figure S2A–S2D). None of our maco-1 alleles were unambiguously mutants. However the premature stop codon associated with the db9 allele would be expected to cause nonsense mediated degradation of maco-1 mRNA, as well as truncating half the MACO-1 protein, and therefore to be a strong loss-of-function mutation. Consistent with this, the phenotypes of maco-1(db9)/ maco-1(db9) and maco-1(db9)/qDf16 were similar (Figure S1G); qDf16 is a large deletion that spans the maco-1 interval (see Materials and Methods). cDNA analyses indicated that the D2092.5 gene can encode two splice isoforms by alternate splicing at exon 5 (Figure 2A). The proteins encoded by the resulting mRNAs were 908 (MACO-1a) and 897 (MACO-1b) amino acids long. Blast searches identified these proteins as homologues of vertebrate macoilins. Reciprocal- ly, searching the C. elegans genome with vertebrate macoilins identified only one homologue, D2092.5 (Figure 2D). At least one macoilin can be found in every animal genome sequenced so far (Figure 2B, 2C). In some fish lineages but not in other vertebrates, a second MACO homologue can be found (MACO-2); MACO-2 probably arose during the genome duplication event that is thought to have occurred in teleost fish. Despite this ubiquity, little PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001341