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

presence of BFA only (Fig. 4 e) , in 41% of the cells man II had a perinuclear localization and in 97 % of the cells it was found only or partly in the ER. As the chase was continued to 180 min at 20~ in the presence of 5 #g/ml of BFA (Fig. 4 g) , man II was still found in 47 % of the cells in perinuclear elements and in 97 % of the cells only or partly in the ER. When the cells were treated with 5/zg/ml of BFA supplemented with 10 mM caffeine at 20~ for 60 min (Fig. 4 d) , man II was found in the ER in 68 % of the cells and in the perinuclear localization in 92 % of the cells. After a 120-min chase in the presence of caffeine, man II was located partly or completely in the ER in 91% of the cells and was perinuclearly located in 75 % of the cells (Fig. 4 f ) . When the chase was carried out for 180 min (Fig. 4 h) , man II was found in the ER in 96 % of the cells and perinuclearly located in 76 % of the cells. These results show that, after 60 min in the presence of caffeine, 5 #g/ml of BFA is able to translocate man II to the ER 0.76-fold slower compared with control cells. After 120 rain, however, there is no significant difference between caffeine-treated and control cells in the number of cells showing label in the ER. However, in caffeinetreated cells the disappearance of the perinuclear labeling of Figure 5 . Caffeine-induced translocation of p58 to the periphery of the cells. BHK-21 cells were grown for 30 min at 20"C in the presence (a and b) or absence (c and d) of 10 mM caffeine, fixed, and double-labeled with antibodies to p58 (a and c) and man II (b and d). The distribution of p58 can be seen to change dramatically after a caffeine treatment at 20"C. In a, p58 can be found scattered through the cytoplasma in contrast to control cells at 20~ (c), where p58 is concentrated in the perinuclear region of the cell in addition to some labeling in peripheral vesicles. When the localization of p58 is compared with the localization of man II in caffeine-treated and in control cells incubated at 20~ p58 can be seen to colocalize only randomly with man II in caffeine-treated cells (a for p58 and b for man II), but to colocalize a great deal in control cells (c for p58 and d for man II). Also, the localization of man II seems not to be affected by the caffeine treatment at 20~ (b). Bar, 20 #m. man II seems to occur more slowly than in the control cells at the 60-min time point by a factor of 1.5, at the 120-min time point by a factor of 1.8, and at the 180-min time point by a factor of 1.6. Thus, these results indicate that caffeine does not inhibit the effect of BFA, but it does appear to slow it down somewhat. Interestingly, it seems that during a 180min chase at 20~ both in caffeine-treated and in control cells the BFA-induced translocation of man II reaches a steady-state situation after a 120-min incubation, whereafter no significant chances in the distribution of man II in the cells seems to occur.