Fig. 1

Putative structure and intracellular localization of SLC35B1. a Protein sequences are from UniProt or GeneBank and shown in single letter code for Homo sapiens (Hs, P78383.1; NM_005827.1), Mus musculus (Mm, P97858.1), Caenorhabditis elegans (Ce, CAC35849), Schizosaccharomyces pombe (Sp, CAB46704.1), Saccharomyces cerevisiae (Sc, CAA97965), Arabidopsis thaliana (At, At1g14360 and At2g02810), and Starkeya novella (YddG, gi:502932551). The sequences were aligned using ClustalX and GeneDoc. The amino and carboxy termini face the cyosol, the double lysine motif near the carboxy terminus of mammalian SLC35B1 serves as ER retention motif. The predicted IQ motif, unique to mammalian SLC35B1, is shown in purple, positively charged clusters in red. SLC35B1/Isoform 2 comprises an amino-terminal extension of 37 amino acids (MRPLPPVGDVRLWTSPPPPLLPVPVVSGSPVGSSGRL) (NM_005827.2), in transcript variant 2 (NM_001278784.1) the first 78 amino acids, including two N-terminal transmembrane helices, of SLC35B1 are replaced by the oligopeptide: MCDQCCVCQDL. b Hypothetical structural model of human SLC35B1, as predicted by the Phyre2 server³⁴. Transmembrane helices 2 (green) plus 3 (blue) and the connecting loop (purple) with the putative IQ motif are highlighted, as are clusters of positively charged amino acid residues (red). c A 4% digitonin extract of canine pancreatic rough microsomal membrane proteins (derived from 6 mg microsomal protein) was subjected to SDS-PAGE in parallel to E. coli membranes (25 µg protein), which were derived from non-transfected and SLC35B1-expressing or SLC35B1/isoform 2-expressing cells. The Western blot was decorated with SLC35B1-specific antibody, validated in Supplementary Fig. 1a, and visualized with peroxidase-coupled secondary antibodies, Super Signal West Pico, and luminescence imaging. Molecular mass standard (M) was run in parallel and electronically copied from the stained blot to the Western blot. The relevant part of the blot is shown; the complete blot is shown in Supplementary Fig. 1b. d HeLa cells were transfected with an expression plasmid encoding SLC35B1-GFP for 8 h, the nuclei were stained with DAPI, and the ER was visualized with Sec62-specific antibody plus Alexa-Fluor-594-coupled secondary antibody and subjected to fluorescence imaging using a super-resolution Elyra microscope³⁸. Representative images and merged images are shown (scale bar 10 µm). Related Western blots are shwon in Supplementary Fig. 1c, d