Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology

Abstract

The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for protein folding, lipid synthesis, and calcium homeostasis. Maintenance of ER structural integrity is crucial for proper function, but much remains to be learned about the molecular players involved. To identify proteins that support the structure of the ER, we performed a proteomic screen and identified nodal modulator (NOMO), a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We found that overexpression of NOMO1 imposes a sheet morphology on the ER, whereas depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network positive for the lysosomal marker lysosomal-associated protein 1. In addition, the levels of key players of autophagy including microtubule-associated protein light chain 3 and autophagy cargo receptor p62/sequestosome 1 strongly increase upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a "beads on a string" structure likely representing consecutive immunoglobulin-like domains. Extending NOMO1 by insertion of additional immunoglobulin folds results in a correlative increase in the ER intermembrane distance. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.

Keywords: cell biology; endoplasmic reticulum; imaging; membrane protein; single-particle analysis; structural model.

Figure 1

Identification of NOMO1 as an abundant and conserved ER-resident protein.A, cells expressing ER-APEX2 were treated with biotin-phenol in absence or presence of hydrogen peroxide, lysed, and subjected to Western blotting using streptavidin-HRP. B, table of top-10 most abundant proteins from MS analysis in order of spectral count; % coverage is the sequence coverage of the protein based on the peptide sequences identified. C, phylogenetic tree of NOMO1 homologs in indicated metazoan and plant species. D, FLAG-NOMO1 domain structure. Note that the FLAG tag was inserted between the cleavable signal sequence (SS) and the luminal domain. CYT, cytosolic tail; ER, endoplasmic reticulum; NOMO1, nodal modulator 1; TM, transmembrane domain.

Figure 2

NOMO depletion results in profound changes of ER morphology.A, confocal images of U2OS cells transfected with the respective siRNA for 48 h. Protein disulfide isomerase (PDI) is used as the ER marker. B, quantification of mRNA level of each NOMO siRNA by qPCR. C, representative image of the phenotypic rescue of the NOMO knockdown phenotype by an siRNA-resistant construct, FLAG-NOMO1r. Calnexin is used as the ER marker. D, quantification of rescuing ability of FLAG-NOMO1r, n = 100, N = 3, p < 0.05. The asterisks denote p < 0.005 compared with the control. Error bars indicate SD. All scale bars are 10 μm. ER, endoplasmic reticulum; NOMO1, nodal modulator 1.

Figure 3

Epistasis analysis of known ER-shaping proteins and NOMO1.A, U2OS cells were treated with respective siRNA for 48 h and stained with PDI as an ER marker. B, representative image of Atl2-FLAG overexpression (left panel) rescuing the NOMO KD phenotype as judged by PDI staining (right panel). C, quantification of the ability of ER-shaping proteins to rescue the NOMO KD ER phenotype, n = 100, N = 4. Error bars indicate SD. ∗∗∗p < 0.002; ∗∗∗∗p < 0.0001. D, representative image of FLAG-NOMO1r overexpression (left panel) rescuing Atl2 KD phenotype (right panel). E, quantification of NOMO1 and Climp63 overexpression rescuing the Atl2 KD phenotype, n = 100, N = 3. Error bars indicate SD. ∗∗p < 0.01. Scale bars are 10 μm. ER, endoplasmic reticulum; NOMO1, nodal modulator 1; ns, not significant; PDI, protein disulfide isomerase.

Figure 4

EM analysis of NOMO and Atl2-depleted cells.A, U2OS cells were successively treated with two doses of siNOMO 24 h apart and fixed 48 h after the second dose for EM processing. The white square in the top panel identifies selection for the middle panel. Asterisks in bottom panel denote free membrane ends. B, U2OS cells successively treated with two doses of siAtl2 as described in panel A. The arrow in second panel indicates an identified membrane outlining the hole. EM, electron microscopy; NOMO, nodal modulator.

Figure 5

Sheet disruption increases autophagy.A, representative images of U2OS cells treated with the respective siRNA to identify lysosome localization using LAMP1 as a marker. The scale bar is 10 μm. B, immunoblot with calnexin and LC3 antibodies using U2OS cells extracts treated with the indicated siRNA. C, immunoblot with BiP and LC3 antibodies of the Baf A-treated samples under the respective siRNA conditions. D, immunoblot using p62, GAPDH, and LC3 antibodies of the Baf A-treated samples under the respective siRNA conditions. E, quantification of the fold change in p62 levels calculated by dividing p62 intensity by GAPDH intensity and normalizing to the untreated control sample. ∗p < 0.05; ∗∗p < 0.01. Baf A, bafilomycin A; LAMP1, lysosomal-associated protein 1.

Figure 6

NOMO1 restricts the lumen of the ER.A, increasing expression of FLAG-NOMO1 in U2OS cells using PDI as the ER marker. All scale bars are 10 μm. B, HeLa cells transfected with the empty vector pcDNA3 as a control. The white box in the top panel identifies selected zoomed ER membrane area in the bottom panel. C, HeLa cells transfected with FLAG-NOMO1. The white box in top panel identifies selected zoomed ER membrane area in bottom panel. D, intermembrane space of HeLa cell ER and NE from panels B and C. Asterisks indicate p < 0.0001. E, intermembrane space of ER and NE cross-sections of U2OS cells treated with the respective siRNAs. Asterisks indicate p < 0.0001. ER, endoplasmic reticulum; NE, nuclear envelope; NOMO1, nodal modulator 1; ns, not significant; PDI, protein disulfide isomerase.

Figure 7

Determination of the NOMO1 oligomeric state.A, elution profile of NOMO1-FLAG on a S200 column. Insert, SDS-PAGE/silver stain of NOMO1-FLAG fraction obtained from preparative SEC. B, in-line SEC-MALS profile of NOMO1-FLAG subjected to a Superose 6 column. The dashed line is the UV trace, molar mass represents the total molar mass of the PDC, protein molar mass is the corrected molar mass to remove contribution of detergent. C, SEC-MALS profile of NOMO^LD-FLAG subjected to a Superose 6 column; lines as defined in panel B. D, in-line SEC-MALS analysis of MBP-TM-CYT subjected to a S200 Increase column. CYT, cytosolic tail; NOMO1, nodal modulator 1; SEC-MALS, size-exclusion chromatography linked to multiangle light scattering; TM, transmembrane.

Figure 8

Single-particle analysis of NOMO1.A, top-10 two-dimensional class averages of ∼7000 picked negative-stain NOMO1-FLAG particles, numbers of particles per class in squares. Mask diameter is 40 nm. A construct layout is inserted to clarify protein domains. B, three-dimensional reconstruction from panel A; the scale bar is 10 nm. C, top-10 two-dimensional class averages of ∼10,000 picked negative-stain NOMO^LD-FLAG particles, the number of particles per class in squares. Mask diameter is 30 nm. A construct layout is inserted. D, three-dimensional reconstruction from panel C, the scale bar is 10 nm. CYT, cytosolic tail; NOMO, nodal modulator; TM, transmembrane domain.

Figure 9

Extended NOMO constructs increase the ER intermembrane space.A, design of the two extended NOMO constructs (see the text for details). B, detection of the extended constructs via SDS-PAGE/immunoblotting using a FLAG antibody. C, validation of ER localization by immunofluorescence. PDI is used as the ER marker. All scale bars are 10 μm. D, representative EM images of HeLa cells expressing the respective construct. E, quantification of the ER intermembrane space for each transfected construct compared with NOMO1-FLAG and control cells. ∗∗∗∗p < 0.0001. F, models for membrane spacing by NOMO1. I, the purple block represents an unknown/speculative interaction partner. II, NOMO interacts with the membrane. III, NOMO forms an antiparallel dimer. ER, endoplasmic reticulum; NOMO1, nodal modulator 1; PDI, protein disulfide isomerase.