JCS03427 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Ribbon representation of the three-dimensional structure of Onco (PDB ID 1ONC). The location of the active site residues (green) and the Ser72Cys mutation (red) that was introduced for fluorescent labeling is indicated. This figure was drawn with PyMOL (http://pymol.sourceforge.net).

• Supplemental Figure 2 -

  Fig. S2. Onco accumulates in recycling endosomes. HeLa cells were transiently transfected with EGFP-tagged Rab4, Rab5 or Rab11. After 24 hours, Onco-Red and Tf-Cy5 were added for 45 minutes. Cells were then fixed and examined by confocal microscopy. Median optical sections; bar, 20 μm. Colocalization quantitations were performed using Metamorph software (Molecular Devices). A minimum of 10 images (n>20 cells) were used for each quantitation. Onco colocalization with Rab11 (60%) was more pronounced than with Rab4 (51%) or Rab5 (48%).

• Supplemental Figure 3 -

  Fig. S3. Onco routing to recycling endosomes is not affected by Baf. HeLa cells were incubated for 45 minutes with Onco-Red and Tf-Cy5 (to label early endosomes) in the presence of 100 nM Baf. When indicated, cells were also labeled with dextran-FITC, a tracer that is addressed to lysosomes. Cells were then processed for immunodetection of markers for late endosome/lysosomes (Lamp-1, Lamp-2 or LBPA), the ER (calnexin) or the trans-Golgi network (TGN46), before mounting and observation under a confocal microscope. Median optical sections; bar, 10 μm. Boxed areas in LBPA and Lamp-2 images indicate regions enlarged on the right. Images obtained from control cells are presented in Fig. 2.

• Supplemental Figure 4 -

  Fig. S4. Translocation efficiency as a function of time. Endosomes labeled with ¹²⁵I-Tf or ¹²⁵I-Onco were incubated for the indicated period of time at 37°C in translocation buffer containing 400 nM Baf and 10 mM ATP. Endosomes were then separated from the medium by ultracentrifugation on a sucrose cushion, before counting the supernatant (SN) and pellet (Pe). Approximately 15% of endosomal Onco translocated in 1 hour, whereas no significant Tf release from the endosomes was observed.

• Supplemental Figure 5 -

  Fig. S5. Onco is released from its receptor(s) at low pH. Jurkat cells were labeled with ¹²⁵I-Onco (or ¹²⁵I-Tf as a control) on ice before washing away excess ligand and incubation on ice in buffered saline adjusted to the indicated pH value. Cells were then spun before counting the supernatant (SN) and pellet (Pe).