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

Since caffeine at 20~ inhibited the ER-to-Golgi membrane traffic, it was of great interest to study whether 10 mM caffeine would also inhibit the Golgi-to-ER traffic. Recently, the fungal antibiotic BFA has been used to study the retrograde transport mechanism from the Golgi complex to the ER (Doms et al., 1989; Lippincott-Schwartz et al., 1989 , 1991a Fujiwara et al., 1988) . We therefore studied the effect of 10 m M caffeine on the BFA-induced redistribution of Golgi membranes at 20~ using man II as a marker. BHK-21 cells were treated for 60, 120, or 180 min with 5 #g/ml of BFA and 50 #g/ml of cycloheximide at 20~ in the presence or absence of 10 m M caffeine. In Fig. 4 a the distribution of the Golgi marker man II is shown before any drug treatments. When the cells were treated with 5/zg/ml of BFA for 90 min at 37~ a characteristic ER staining pattern was observed (Fig. 4 b) . When the cells were treated with 5 #g/ ml of BFA for 60 rain at 20~ (Fig. 4 c) in 60% of the cells man II was found in a perinuclear localization, and in 89 % of the cells, man II was found to be translocated partly or completely to the ER. After a 120-min chase at 20~ in the Figure 4 . The BFA-induced recycling of man II from the Golgi complex to the ER is not inhibited by caffeine at 20"C. BHK-21 cells were incubated with BFA (5 #g/ml) at 37~ (b), with BFA (5 ~g/ml) and 10 mM caffeine at 20~ for 60, 120, and 180 min (d,f, and h, respectively) or with BFA (5/~g/rnl) only at 20"C for 60, 120, and 180 min (c, e, and g, respectively), fixed, and processed for immunofluoreseence microscopy. In all cases 50 /tg/ml of cycloheximide was supplemented to prevent further synthesis of man II. Normal distribution of man II in BHK-21 cells (a). In b man II is observed to label the ER after a 90-rain BFA treatment at 37"C. In caffeinetreated cells at 20~ the BFAinduced transloeation of man II to the ER is not inhibited (d, 60 rain;f, 120 rain; andh, 180 min). When compared with the control cells (c, 60 min; e, 120 rain; and g, 180 rain) the BFA-induced disappearance of the perinuclear Golgi labeling of man II seems to happen more slowly in the caffeinetreated ceils than in the control cells. Bar, 20 ~m.