Fig 1: Subcellular distribution of ROGDI and Rabconnectin-3. A, subcellular fractionation of 4T1 cell lysate. Cells were lysed mechanically by passing through a 27-gauge needle and centrifuged at 10,000g to pellet nuclei and mitochondria. A portion of the resulting postmitochondrial supernatant was reserved as input as described in the Experimental procedures section. The postmitochondrial supernatant was then centrifuged to pellet membranes and supernatant (cytosol). Both the membrane pellet and cytosolic samples were denatured and analyzed by SDS-PAGE and Western blotting with antibodies against the indicated proteins. Molecular mass markers are shown on left. All samples were run on a single SDS-PAGE gel, and immunoblots probed anti-ROGDI and anti-DMXL1 blots were cut at the 50 kDa molecular mass marker. B, a lysosomal fraction was isolated by affinity purification using FLAG-tagged TMEM192 from mechanically lysed 4T1 cells as described in the Experimental procedures section. Cells were transiently transfected with pCDNA3-TMEM192-FLAG-HA, and a postmitochondrial supernatant was generated as in A. A portion of this sample was set aside as input. Lysosomes were isolated by binding to anti-HA nanobody magnetic beads. Input (0.75% of total), sequential washes of the beads (washes 1 and 2), and the final eluted sample (LysoIP) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Samples separated by dotted lines were from the same gel, cut at the position of the line. Intensities of the lysate and LysoIP samples for each sample were determined using FIJI, and the ratio of LysoIP/lysate intensity is shown below the protein label. The data shown are representative of three independent experiments. HA, hemagglutinin; LysoIP, lysosomal immunoprecipitation.
Fig 2: AlphaFold3 model of ROGDI binding to N-terminal (NT) domains of Rabconnectin-3α and β. AlphaFold3 model of DMXL1 NT (amino acids 1–630), WDR7 NT (1–600), and full-length ROGDI. The ipTM for this model is 0.82 and pTM is 0.81, as described in the Experimental procedures section. These reflect a high confidence model. ipTM, interface predicted template modeling; pTM, predicted template modeling.
Fig 3: DMXL1 and ROGDI partially colocalize on perinuclear lysosomes.A, immunofluorescence micrograph of a 4T1 cell costained with indirectly labeled anti-LAMP2 (magenta) and directly labeled anti-ROGDI (green) antibodies. LAMP2 serves as a lysosomal marker. Images are from a single slice of a confocal stack. The composite image with DAPI (blue) shown to the right illustrates extensive overlap between the anti-LAMP2 and anti-ROGDI channels on perinuclear lysosomes. The dotted line indicates the region of interest used for determination of Pearson's value. B, immunofluorescence micrograph of a 4T1 cell costained with indirectly labeled anti-LAMP2 (magenta) and directly labeled anti-DMXL1 (green) antibodies. The composite image to the right shows overlap between the anti-LAMP2 and anti-DMXL1 channels. For both A and B, the Pearson's R value was calculated as described in the Experimental procedures section. DAPI, 4′,6-diamidino-2-phenylindole; LAMP2, lysosome-associated membrane protein 2.
Fig 4: ROGDI partially localizes with V1A on perinuclear lysosomes. A, wide-field immunofluorescence micrographs of 4T1 cells costained with directly labeled anti-V1 A subunit (magenta) and anti-ROGDI (cyan) antibodies as described in the Experimental procedures section. Two lines were drawn through ROGDI puncta both in the peripheral (1) and perinuclear (2) regions. Fluorescence intensity along each line is plotted; ROGDI is orange and V1A is blue. Graph 1 corresponds to peripheral puncta, and graph 2 corresponds to perinuclear puncta. A merged image combining the ROGDI and V1A channels is at the right of the line scan plots. B, immunofluorescence micrograph of 4T1 cells costained with anti-LAMP2 (cyan) antibody and fixable LysoTracker DND-99 (magenta), an indicator of lysosomal acidification. Cells were stained with LysoTracker DND-99 immediately prior to fixation and permeabilization. Permeabilization and fixation were performed as in Figure 5A. Both images are from a wide-field microscope. LAMP2, lysosome-associated membrane protein 2.
Fig 5: ROGDI interacts with conserved β-propeller region of Rav1 and Rabconnecin-3.A, secondary structure comparison of Rabconnectin-3α, Rabconnectin-3β, and Rav1. Regions that are modeled to form β-propellers and α-solenoid are indicated with corresponding amino acid positions. B, fusion proteins of ROGDI, DMXL2 1–625, WDR7 1–596, WDR72 1–589, and Rav1 2–720 were generated in pAS2 and pACT2 plasmids as described in the Experimental procedures section. All pACT2 plasmids were transformed into the PJ69-4α two-hybrid strain, and pAS2 plasmids were transformed into PJ69-4a yeast two-hybrid strain. Diploids were selected on SD media lacking leucine and tryptophan (SD- Trp, Leu). Growth on test media lacking tryptophan, leucine, histidine, and adenine (SD- Trp, Leu, Ade, His) is shown and indicates protein–protein interactions between the fusion proteins. pACT2 vector only and pAS1-lamin were used as negative controls.
Supplier Page from Abcam for Anti-ROGDI antibody [EPR15190]