Figure 7.
The GM130 peptide releases COPI vesicles from Golgi membranes. Salt-washed Golgi membranes were incubated with interphase cytosol for 30 min at 37°C in the presence of GTPγS. In some experiments, the cytosol was preincubated for 15 min on ice with the GM130 peptide at the indicated molar ratios over cytosolic p115. At the end of the incubation, those samples using untreated cytosol were either left untreated (−salt; GM130 peptide) or treated with KCl at a final concentration of 250 mM to release COPI vesicles (+salt). Golgi remnants were sedimented in a microfuge and released COPI vesicles in the supernatant were recovered by high speed centrifugation through a 35% sucrose cushion. Remnants and vesicles were fractionated by SDS-PAGE and quantitated by Western blotting using antibodies to β-COP and p115. (A) Release of COPI vesicles is expressed as a percentage of the +salt control ± SEM (n = 3). (B) p115 bound to Golgi remnants is expressed as a percentage of the −salt control ± SEM (n = 3).