Fig. 4. Strong interaction of GAPR-1 with Golgi membranes. In all panels, the incubations were analysed by SDS-PAGE and western blotting for the presence of the indicated proteins. (A) CHO cells were incubated for 30 minutes in the absence (lane 2 and 3) or presence of 5 µM Brefeldin A (lane 4 and 5). The BFA-induced redistribution of GAPR-1 into tubulo-vesicular structures was confirmed by concomitant immunofluorescence (data not shown). After homogenisation, the homogenate was centrifuged for 1hour at 100,000 g to yield a total membrane (lanes 2 and 4) and cytosolic fraction (lanes 3 and 5). GAPR-1 was immunoprecipitated from the membrane fraction (2 mg) or from the cytosolic fraction (2 mg) as described in the Materials and Methods. As a control, GAPR-1 was immunoprecipitated from isolated CHO Golgi membranes (50 µg) (lane 1). (B) 50 µg of CHO Golgi membranes (lanes 1-3) was incubated with 1 M KCl (lane 2) or with 0.1 M Na₂CO₃, pH 11 (lane 3) for 30 minutes on ice. After centrifugation through a 15% (w/v) sucrose cushion, equal amounts of membrane (29 nmol phospholipid) were analysed. (C) CHO Golgi membranes (25 µg) were incubated for 30 minues at 4°C in the absence (lane 1) or presence (lanes 2 an 3) of 3 µl of bacterially expressed, purified and non-myristoylated GAPR-1 (5.3 mg/ml) in 25 mM Hepes/KOH, pH 7.2, 20 mM Kcl, 2.5 mM magnesium acetate, 0.1 M sucrose, 1 mg/ml ovalbumine and 10 mM DTT. KCl (1 M final concentration) was added to one incubation (lane 3). Golgi membranes were re-isolated by centrifugation through a 15% (w/v) sucrose cushion. (D) CHO Golgi membranes (50 µg) were incubated with Hydroxylsulfosuccinimidyl-4-azidobenzoate (5 mM) in PBS for 30 minutes at RT and left on ice (lane 1) or irradiated for 10 minutes at 254 nm (lane 2 and 3) and analysed for crosslinked products. For immunoprecipitation (lane 3), 500 µg of Golgi membranes was used, and GAPR-1 was immunoprecipitated as described in Materials and Methods.

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