UEV-1 and Glutamate Receptors Figure 3. UEV-1 is broadly expressed. (A–F) Fluorescence from animals transgenic for Puev-1::uev-1::gfp. Expression is detected in (A) pharynx and multiple head neurons, (B) distal tip cell, vulval epithelia, and ventral cord motoneurons (arrowheads), (C) most cells of embryos (gastrulating embryo shown from ventral view), (D) intestinal epithelia and motoneurons (arrowheads), (E) body wall muscle, and (F) ventral cord motoneurons. (D–F) Arrows indicate nuclear enrichment of the UEV-1::GFP protein. (G–L) Fluorescence from uev-1(od10)-mutant animals co-expressing (G, J) GLR-1::GFP and (H, K) mCherry::UEV-1 via the glr-1 promoter. (I, L) Merged images. (G–I) UEV-1 is enriched in nuclei (arrow), but can be found at punctate structures in the cell body cytoplasm (PVC neuron cell body is shown). (J–L) UEV-1 is uniformly distributed along ventral cord neurites. Bar, 5 microns. doi:10.1371/journal.pone.0014291.g003 We reasoned that if UEV-1 regulates GLR-1 as part of ERAD, then in the absence of both UEV-1 and UPR function, GLR-1 receptors that would normally have been degraded would instead accumulate to higher levels in the ER. To address this question, we captured fluorescent images of cell bodies from wild type, uev-1 single mutants, ire-1 single mutants, and uev-1 ire-1 double mutants. While we could not find a single microscope setting with the dynamic range to quantify all four genotypes relative to each other, we were nevertheless able to compare uev-1 single mutants to wild type, and uev-1 ire-1 double mutants to ire-1 single mutants. We found that, unlike uev-1 single mutants, which have elevated cell body levels of GLR-1 relative to wild type (Figure 5C, K), instead uev-1 ire-1 double mutants have slightly decreased levels of GLR-1 relative to ire-1 single mutants (Figure 5G, L). Our results suggest that mutations like ire-1 that trap GLR-1 receptors in the ER preclude those receptors from regulation by UEV-1, suggesting that UEV-1 regulates GLR-1 trafficking at a step after the ER, making it unlikely that UEV-1 is part of the ERAD pathway. An alternative possibility for how UEV-1 regulates GLR-1 is that it regulates trafficking at a step further down in the secretion pathway, perhaps by mediating GLR-1 turnover after endocytosis. Indeed, when we analyzed the fluorescence levels of GLR-1::GFP in profile along the ventral cord, we found that the unlocalized PLoS ONE | www.plosone.org 6 December 2010 | Volume 5 | Issue 12 | e14291