Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

It has been shown that the pre-Golgi markers p58 in normal rat kidney cells (Saraste and Svensson, 1991) and p53 (Schweizer et al., 1988) in M1 cells ) get translocated to the periphery of the cell after a BFA treatment. The distribution and morphology of p58positive elements in BHK-21 cells after incubation at 20~ in the presence of 10 mM caffeine resembled that observed earlier in BFA-treated cells. Even though incubation at 20~ in the presence of caffeine did not result in the movement of a resident Golgi stack marker to the ER (see Fig. 5 b) , it did cause profound effects on the organization of the Golgi complex (see Fig. 7 a) . ~COP, a protein known to be associated with the pre-Golgi and the Golgi stack membranes (Duden et al., 1991) is released rapidly from the Golgi membranes in cells treated with BFA . We therefore tested whether caffeine would have an effect on the localization of E-COP. BHK-21 cells were incubated at 20~ in the presence or absence of 10 mM caffeine, fixed, and processed for immunofluorescence microscopy. In Fig. 9 a, the distribution of E-COP is shown in cells before any treatments. ~COP has a strong perinuclear localization and also the labeling of cytoplasmic vesicular elements is observed (Fig. 9 a) . When cells were incubated at 20~ for 60 min in the absence of caffeine (Fig. 9 b) , the distribution of /~-COP was essentially the same as in control cells without any treatments (Fig. 9 a) . A 60-min incubation at 20~ with 10 mM caffeine did not have any detectable effect on the distribution of/~-COP (Fig. 9 c) . Because of the strong perinuclear labeling of E-COP, possible changes in the distribution of pre-and cis-Golgi elements were undetectable in the experiment. This result nevertheless suggests that BFA and caffeine affect the membranes of the Golgi complex through different mechanisms.