Figure 3.

GAIP in carrier vesicle fractions has GAP activity. (A) GAIP is enriched in carrier vesicle and residual microsome fractions. Each fraction indicated (50 μg) was separated by SDS/12% PAGE and immunoblotted with anti-GAIP (N) followed by [¹²⁵I]protein A. H, homogenate; PNS, postnuclear supernatant; TM, total microsomes; Cyt, cytosol; GL, Golgi light fraction; GH, Golgi heavy fraction; CV1, carrier vesicle 1 fraction; CV2, carrier vesicle 2 fraction; RM, residual microsomes. (B) Time course of GAP activity. GAP activity was evaluated over time after addition of 1 μg of each fraction to [γ-³²P]GTP-loaded Gα_i3. The CV2 fraction (▵) has GAP activity. In contrast, the homogenate (□) and the boiled CV2 fraction (♦) show no GAP activity. (C) Dose dependence of GAP activity. Different amounts of CV2 were added to [γ-³²P]GTP-loaded Gα_i3. Reactions were stopped after 2 (▴) and 5 (●) min of hydrolysis. Results are obtained after subtraction of the estimated background (value at zero time point). GAP activity of the CV2 fraction is a linear function of the amount added in the reaction. Data shown are representative of three experiments.