Fig. 2. GAPR-1 is a Golgi-localized peripheral membrane protein. (A) The enrichment of GAPR-1 in CHO Golgi membranes and CHO GIC was determined by comparing the amounts of GAPR-1 in CHO homogenate (lane 1, 50 µg), CHO cytosol (lane 2, 50 µg), CHO Golgi membranes (lane 3, 5 µg) and CHO GIC (lane 4, 0.5 µg) by use of SDS-PAGE and western blotting. (B) The topology of GAPR-1 at Golgi membranes was determined by protease digestion of Golgi membranes (lanes 2-4) in the absence or presence of Trypsin inhibitor (lane 3) or Triton X-100 (lane 4). After incubation (as described in Materials and Methods), the proteins were separated by SDS-PAGE and GAPR-1 and p23 were visualized by western-blotting. (C) Exclusion of GAPR-1 from COPI-coated vesicles. Equivalent amounts of isolated Golgi membranes and COPI-coated vesicles (10.8 µg total phospholipid) were analysed by SDS-PAGE and western blotting for the presence of GAPR-1. As positive controls for COPI-coated vesicles, the blots were probed with antibodies against ß-COP and p23. Golgi membranes and COPI-coated vesicles were isolated as described in Materials and Methods.

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