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

As markers for Golgi membranes we used p58 protein, known to be localized in various cell types in the pre-Golgi elements and in cis-Golgi (Saraste et al., 1987; Saraste and Svensson 1991; Lahtinen et al., 1992) , and a 135-kD integral Golgi stack membrane protein Burke et al., 1982) which has recently been shown to be identical with man II (Baron and Garoff, 1990) . BHK-21 cells were treated for 30, 60, or 120 min with or without 10 mM caffeine at 20~ fixed, and labeled with a polyclonal antibody to p58 and a monoclonal antibody to man II (135 kD). Surprisingly, after 30 min at 20~ in the presence of Figure 6 . The caffeine-induced translocation of p58 to the periphery of the cells is reversible. BHK-21 cells were incubated in the presence (20~ caffeine) or absence (20 ~ C control) of 10 mM caffeine at 20~ for 60 rain. Cells incubated at 20~ with caffeine were further incubated 60 rain without caffeine at 20~ (reversion), fixed, and doubled-labeled with antibodies to p58 and man II. no drug shows the normal distribution of p58 and man II in BHK-21 cells. In reversion, the perinuclear localization of p58, similar to that in 20~ control, is restored. Bar, 20 #m. caffeine, p58 was found scattered throughout the cytoplasm (Fig. 5 a) whereas man II still remained concentrated in a perinuclear localization (Fig. 5 b) . Incubation for an additional 30 or 90 min showed similar distribution of p58 and man II (data not shown) (see also Fig. 6 for a 60-rain incubation). In control cells, incubated for 30 rain at 20~ p58 and man I1 (Fig. 5, c and d, respectively) were found to colocalize largely, except for the peripheral vesicles positive only for p58. Fig. 5 , a and c, demonstrates that p58 is translocated away from its normal perinuclear localization at 20~ in the presence of 10 mM caffeine. In caffeine-treated cells, the colocalization of p58 (Fig. 5 a) and man II (Fig. 5 b) oc-curred only rarely. Even though the majority of the p58 protein seemed to move to the periphery of the cell in the presence of caffeine at 20~ some p58-positive vesicles still colocalized with the perinuclear elements also labeled with antibodies to man II. This could partly be due to an apparent colocalization of not the same but closely adjacent structures. These results suggest that 10 mM caffeine at 20~ induces a translocation of p58-positive, pre-Golgi, and cis-Golgi membranes to the periphery of the cells leaving man II-positive membranes perinuclearly located.