In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na+K+-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, we found that dPob/EMC3 deficiency induces rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration.DOI: http://dx.doi.org/10.7554/eLife.06306.001

Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.

SUMMARYSorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, we screened mutants in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. We found that deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na + K + -ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. Our results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency.

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2 ⁄ Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-b and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Recent research has revealed that the maternal non-coding RNA genes (Gtl2, Rian and Mirg) from the Dlk1-Dio3 imprinted cluster are closely related to the full development potential of the induced pluripotent stem cells (iPSCs). Transcriptional silencing of these genes failed to generate all-iPSC mice, indicating their significant contribution to embryogenesis. However, except for Gtl2, little information regarding these genes has been acquired in this cluster. In the present study, we analyzed the spatiotemporal expression patterns of Mirg during mouse embryogenesis. Using in situ hybridization and quantitative PCR, we demonstrated that Mirg non-coding RNA exhibited sustained expression throughout mouse embryogenesis from E8.5 to E18.5. Strong expression was detected in the central nervous system (E9.5-E15.5) and various skeletal muscles (E13.5 and E15.5), and the subcellular localization appeared to be in the nuclei. The pituitary and adrenal gland also showed high expression of Mirg, but, unlike the skeletal muscles and the neural circuitry, the signals were not concentrated in the nuclei. In the major internal organs, Mirg maintained low expression during embryogenesis (E12.5-E18.5) whereas in the liver and the developing lung, Mirg was expressed with a gradually decreasing trend and a gradually raising trend, respectively. These findings indicate that temporal regulation of Mirg expression may be required during specific stages and in specific tissues during embryonic development.

Sphingolipid metabolic dysregulation has increasingly been considered to be a drug-resistance mechanism for a variety of tumors. In this study, through an LC–MS assay, LIM and SH3 protein 1 (LASP1) was identified as a sphingolipid-metabolism-involved protein, and short-chain enoyl-CoA hydratase (ECHS1) was identified as a new LASP1-interacting protein through a protein assay in colorectal cancer (CRC). Gain- and loss-of-function analyses demonstrated the stimulatory role played by ECHS1 in CRC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies of the underlying tumor-supportive oncometabolism indicate that ECHS1 enables altering ceramide (Cer) metabolism that increases glycosphingolipid synthesis (HexCer) by promoting UDP-glucose ceramide glycosyltransferase (UGCG). Further analysis showed that ECHS1 promotes CRC progression and drug resistance by releasing reactive oxygen species (ROS) and interfering mitochondrial membrane potential via the PI3K/Akt/mTOR-dependent signaling pathway. Meanwhile, the phenomenon of promoting the survival and drug resistance of CRC cells caused by ECHS1 could be reversed by Eliglustat, a specific inhibitor of UCCG, in vitro and in vivo. IHC assay showed that ECHS1 was overexpressed in CRC tissues, which was related to the differentiation and poor prognosis of CRC patients. This study provides new insight into the mechanism by which phospholipids promote drug resistance in CRC and identifies potential targets for future therapies.

Metabolic reprogramming has been shown to be involved in cancer-induced pre-metastatic niche (PMN) formation, but the underlying mechanisms have been insufficiently explored. Here, we showed that hydroxyacid oxidase 1 (HAO1), a rate-limiting enzyme of oxalate synthesis, was upregulated in the alveolar epithelial cells of mice bearing metastatic breast cancer cells at the pre-metastatic stage, leading to oxalate accumulation in lung tissue. Lung oxalate accumulation induced neutrophil extracellular trap (NET) formation by activating NADPH oxidase, which facilitated the formation of pre-metastatic niche. In addition, lung oxalate accumulation promoted the proliferation of metastatic cancer cells by activating the MAPK signaling pathway. Pharmacologic inhibition of HAO1 could effectively suppress the lung oxalate accumulation induced by primary cancer, consequently dampening lung metastasis of breast cancer. Breast cancer cells induced HAO1 expression and oxalate accumulation in alveolar epithelial cells by activating TLR3-IRF3 signaling. Collectively, these findings underscore the role of HAO1-mediated oxalate metabolism in cancer-induced lung PMN formation and metastasis. HAO1 could be an appealing therapeutic target for preventing lung metastasis of cancer.

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