Macoilin: an ER Protein Regulating Neuron Activity Figure 2. maco-1 mutations disrupt macoilin, a polytopic membrane protein with coiled-coil domains that is conserved across Eumetazoa. (A) maco-1 mutations disrupt D2092.5 which encodes C. elegans macoilin. Arrows indicate locations of the three maco-1 alleles; also indicated is the alternative splicing site. (B–C) Unrooted Neighbor-Joining tree of MACO-1 homologues and 10000 bootstrap replicates analysis and scale bar denoting 0.10 and 0.25 changes per site in (B) and (C), respectively. (B) Branches are grouped by phyla with colored lobes; the average sequence identity with MACO-1 is shown in percentile figures. (C) Zoom-in of the tree of the vertebrate sub-phyla. MACO-2 is a second macoilin gene found in some fish lineages but not in other vertebrates (see text). (D) Multiple alignments of MACO-1 homologues. Predicted transmembrane (TM) and coiled-coil (CC) domains in C. elegans and H. sapiens are shown in red and blue respectively. CE, C. elegans; DM, D. melanogaster; NV, N. vectensis; TR; HS, H. sapiens. A more extensive alignment can be found in Figure S10. doi:10.1371/journal.pgen.1001341.g002 (Figure 4N). Both transgenic and endogenous MACO-1 was also found restricted to neuronal cell bodies at embryonic develop- mental stages (Figure S5). The striking localization of MACO-1 to the cell body raised the possibility that it resides in a specific membrane compart- ment. To investigate this we created different transgenic lines that expressed organelle-specific markers in a subset of neurons, using the glr-1 promoter (glr = glutamate receptor) (Figure 4E–4M; Figure S6). The markers used (a kind gift of M. M. Rolls, Pennsylvania State University) were phosphatidylinositol synthase (PIS) for the general endoplasmic reticulum (ER), translocating chain-associating membrane protein (TRAM) for rough ER, Emerin for the nuclear envelope, and Mannosidase (MANS) for the Golgi. All markers were tagged at the N-terminus with YFP. Co-immunostaining of MACO-1 and YFP revealed partial co- localization between MACO-1 and the ER general marker, YFP- PIS (Figure 4E–4I). Within the ER, MACO-1 was further co- localized with a marker restricted to the rough ER, YFP-TRAM, and nuclear envelope, YFP-Emerin (Figure 4J-4M and Figure S6A–S6D). Similar ER localization was seen in embryonic stages (Figure S6). No significant co-localization was observed with the Golgi-specific marker, YFP-MANS (Figure S6E–S6H; however the cell bodies of C. elegans neurons are small (2 microns), making it difficult to exclude the possibility that there is a low amount of MACO-1 in Golgi. These co-localization experiments suggest that MACO-1 predominantly resides in the ER of neurons, in particular in rough ER and nuclear envelope. We observed no gross morphological defects in maco-1 worms in any of the sub- cellular compartments expressing the YFP tagged constructs described above (data not shown). PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001341