Fig 2: SLC35B1 knockdown in HeLa cells does not dramatically affect cell growth, cell and ER morphology. a Quantitative RT-PCR performed after the transfection of HeLa cells with control siRNA or with SLC35B1- or SLC35B1-UTR targeting siRNA for 96 h. One set of control siRNA samples was treated with tunicamycin (Tu, 2.5 µg/ml) for 2 h in order to induce UPR40. Data from three independent experiments are reported together with the individual data points as % of control with SEM. The light lines indicate the 50 and 150% values. The HSPA5 gene encodes BiP, the DDIT3 gene CHOP. b HeLa cells were transfected with control siRNA or with SLC35B1-targeting or SLC35B1-UTR-targeting siRNA for 48 h, transferred to xCELLigence plates (5 × 103 cells per well), and cultivated for 100 h. Cell growth was monitored in real-time. c HeLa cells were transfected with control siRNA or with SLC35B1-UTR-targeting siRNA for 96 h, stained with DAPI and with a Sec62-specific antibody, and analyzed on the super-resolution microscope38. Representative images are shown (scale bar 10 µm)

Fig 3: Heterologously expressed SLC35B1 and SLC35B1/Isoform 2 are antiporters for ADP and ATP. a, b After 5 min of uptake of 20 µM [α32P]ATP as described in Fig. 2, efflux was induced by the addition of ten-fold molar excess of unlabeled ATP. Data are reported as the mean of at least three independent experiments and are shown with the standard error of the mean (SEM). See Supplementary Fig. 2 for the demonstration of ATP export into the cell supernatants under similar conditions. c–f Proteoliposomes harboring membrane proteins from SLC35B1 (c, d) or SLC35B1/Isoform 2 (e, f) transfected E. coli cells were prepared as described in Methods. Time dependent accumulation of 50 µM [α32P]-ATP in unloaded (pink) or preloaded proteoliposomes (c, e) or 50 µM [α32P]-ADP in unloaded (light green) or preloaded proteoliposomes (d, f). Proteoliposomes were preloaded with 10 mM ADP (empty rhombs) or 10 mM ATP (filled rhombs). [α32P]-ATP or [α32P]-ADP was present at a concentration of 50 µM. Uptake was terminated after 2 min. Data are reported as the mean of at least three independent experiments and are shown with the standard error of the mean (SEM)

Fig 4: 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

Fig 5: ERAT4.01 reveals ATP depletion in the ER of HeLa cells after SLC35B1 knockdown. a–d HeLa cells were transfected with control siRNA or with SLC35B1- or SLC35B1-UTR-targeting siRNA for 72 h, then transfected with ERAT4.01. After 24 h, they were imaged by fluorescence microscopy. Where indicated, Thapsigargin (Tg, 1 µM) or 2-deoxy-glucose (2-DG, 10 mM) were added. a Images were recorded using transmission light or fluorescence. Representative images are shown (scale bar 10 µm). b Time-resolved live cell recordings of ER luminal ATP levels are shown as FRET-ratio F535/F480. Data are presented as means for ctrl, n = 33 cells, UTR siRNA, n = 20, SLC35B1 siRNA, n = 21, from at least three independent experiments. c Statistical analysis of the resting ATP levels in the experiments shown in b. Three time points before Tg addition were averaged (indicated as 1) and subtracted from the MAX-values (indicated as 2) following Tg addition for each single cell. Data are presented as mean with SEM. The indicated pairs were assessed by unpaired, two-sided standard Student´s t-test (*P < 0.05, **P < 0.01, **P < 0.001). d Statistical analysis of the Tg-induced ATP increase in the experiments shown in b. e, f HeLa cells were transfected with control siRNA or with SLC35B1- or SLC35B1-UTR-targeting siRNA, transfected with ATeam, and imaged. The mean values of time-resolved live cell recordings of cytosolic ATP levels (e) and the corresponding statistical analysis (f) are shown for ctrl, n = 116 cells, UTR siRNA, n = 95, SLC35B1 siRNA, n = 57 from at least three independent experiments. g HeLa cells were transfected with control siRNA or with SLC35B1-targeting or SLC35B1-UTR-targeting siRNA for 96 h, and total cellular ATP was determined using ApoSensor according to the manufacturer´s protocol. Data from three independent experiments are reported together with the individual data points as % of control with SEM