Fig. S1. Time-lapse observations of Golgi-derived transport carriers containing dGGA and Lerp. (A) Schematic representation of mCherry-Lerp-TMC construct. Positions of the signal sequence (ss), transmembrane domain (TM) and cytoplasmic region (cyt) of Lerp are indicated. (B, C, and D) S2 cells transiently expressing EGFP-dGGA and mCherry-Lerp-TMC were subjected to time-lapse microscopy. A typical cell is shown in B, where both proteins colocalize in the large puncta corresponding to the Golgi complex. Images were acquired every 5 seconds at 25C (C). Note that a vesicular structure containing both proteins is emanated from the large Golgi structures (arrowheads). Serial images shown in (C) are projected into a single image to trace the vesicle (arrows). Scale bar: 5 m.

Fig. S2. Characterization of Drosophila AP-1 complex. (A) Expression of AP47, m1 subunit of drosophila AP-1 (dAP-1) in S2 cells. Lysate of S2 cells cultured with (AP47) or without (con) siRNA for AP47, or those stably expressing the AP47 tagged with V5 epitope (AP47-V5; clone mu1-4) was subjected to immunoblotting with anti-AP47 antibody. (B) Subcellular fractionation of AP47. A PNS fraction prepared from the mu1-4 cell was separated to membrane- (M) and cytosol- (C) fractions by ultracentrifugation as described in Materials and Methods. Each fraction was subjected to immunoblotting for dGGA (upper panel), dGM130 (middle panel) or AP47-V5 (lower panel). (C) Gel filtration analysis. The cytosolic fraction from mu1-4 cells was separated through Superdex 200, which were then analyzed by immunoblotting using antibodies to dCHC, dGGA and V5. Molecular size markers with Stokes radii given in Angstroms are indicated. Void fraction is indicated with v. (D) BFA sensitive localization of AP47-V5 in S2. The mu1-4 cells were treated with (+BFA) or without (-BFA) 2 g/ml of BFA for 1 minute, and then subjected to immunofluorescence microscopy with anti-V5 (AP47-V5, green) and anti-p120 (p120, red) antibodies. Scale bar: 10 m.

Fig. S3. Immunodetection of Drosophila Arf1 and GGA. Anti-hARF antibody cross-reacts with Drosophila ARF1. Ten g of S2 or HeLa total cell lysates were subjected to immunoblotting with anti-human ARF antibody. Numbers on the left indicate the positions of molecular mass markers (in kilodaltons).

Movie 1. dGGA positive transport carrier contains mCherry-Lerp. Live imaging of GFP-dGGA and mCherry-Lerp-TMC was carried out in the S2 cells as described in Fig. S3. Images were acquired every 5 seconds at 25°C and the video rate is 3 frames per second.

Movie 2. dGGA on Golgi compartments can be exchanged with its cytosolic fraction. S2 cells stably expressing EGFP-dGGA were subjected to FRAP (fluorescence recovery after photobleaching) analysis. One of the Golgi complex labeled with EGFP-dGGA was photobleached and the fluorescence recovery in the bleached area was imaged every 5 seconds. Quantitative analysis was performed as shown in Fig. 3A. The video rate is 2 frames per second.