Figure 5.

Cytosolic GMx33 associates with stripped membranes in a GTPor ATP-dependent manner. (A) Rat liver Golgi fraction was stripped with 1 M KCl, split into 15 μg aliquots, and incubated with cytosol (800 μg per aliquot) for 30 min with the indicated treatment as described in Materials and Methods. Half of each sample was analyzed by immunoblot with antibodies against the following proteins: β-COP, p115, GMx33, and p24. For densitometry, the GMx33 signal was standardized to the signal for the Golgi-transmembrane protein, p24, and the data are in the bottom part of the panel. (B) Rat liver Golgi fraction was stripped with 2 M NaCl. Cytosolic proteins were incubated with the stripped membrane in the presence of the indicated nucleotides and samples were analyzed as in A. (C) Rat liver Golgi fraction was stripped as in A and treated as indicated with the indicated nucleotides. (D) Rat liver Golgi fraction was not stripped and was subjected to the indicated number of washes at 4°C. Between each wash the fraction was incubated in cytosol buffer for 10 min at 37°C. The final membrane pellets were immunoblotted with antibodies specific for GMx33 and p24. Quantitation was performed as in A and is provided below the blot.