Figure 2.

Figure 2.

ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots of TC-71 lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.