Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1 - GGA1 expression is not affected by GGA2-depletion. HeLa cells were transfected twice with siRNA directed to GGA2 (see Materials and Methods). After the second round of transfection, equivalent amounts of homogenates of siRNA-treated cells (labels on top) were subjected to SDS-PAGE and immunoblotting using antibodies to the indicated proteins (labels on the right).
• Supplementary Video 1 - YFP-GGA1-containing TCs also move from the periphery of the cell to the TGN. H4 cells were transfected twice with siRNAs directed to GAPDH (Mock) and, after the second round of siRNA treatment, were transfected with YFP-GGA1. The mobility of vesicles containing YFP-GGA1 in cells was followed by time-lapse fluorescence microscopy. The upper panels show cells with vesicles moving away from the TGN region (trajectories indicated by green lines), and other vesicles moving toward the TGN region (trajectories indicated by red lines). The lower panels show a two-fold magnification of the region marked with a yellow box in the respective upper panel. Time after the start of imaging (in seconds) is shown on the lower right. Bars, 10 μm (upper panel), 5 μm (lower panel).
• Supplementary Video 2 - Decreased mobility of YFP-GGA1-containing carriers in cells depleted of p56. H4 cells were transfected twice with siRNAs directed to GAPDH (Mock) or p56 and, after the second round of siRNA treatment, were transfected with YFP-GGA1. The effects of the RNAi on the mobility of vesicles containing YFP-GGA1 in cells were assessed by time-lapse fluorescence microscopy. Time after the start of imaging (in seconds) is shown on the lower right. Bar, 10 μm.
• Supplementary Video 3 - Binding of p56 to GGAs is required for the mobility of YFP-GGA1-containing carriers. H4 cells were transfected twice with an siRNA directed to the 5' untranslated region of p56 (p56-5') and, after the second round of siRNA treatment, were co-transfected with YFP-GGA1 and either with Myc-p56 (Myc-epitope-tagged, full-length p56), or Myc-ΔN-p56 (Myc-epitope-tagged construct lacking the N-terminal, GGA-interacting segment of p56; residues 125-441). The effects of the expression of the Myc-epitope-tagged proteins on the mobility of vesicles containing YFP-GGA1 were assessed by time-lapse microscopy. Time after the start of imaging (in seconds) is shown on the lower right. Bar, 10 μm.
• Supplementary Table 1