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

The observed inhibition of ER-exit of SFV glycoproteins could be a result of not only the inhibition of membrane traffic, but also other factors, such as inhibition of proper folding and oligomerization, known to control the ER-exit of individual proteins (Rose and Doms, 1988; Helenius et al., 1992) . To test the effect of 10 mM caffeine and 20~ on the movement of cellular proteins, we followed the recovery of Golgi membranes from BFA treatment using as a marker man II, a resident Golgi membrane protein. BFA has recenrly been shown to cause the redistribution of man II and other resident Golgi proteins as well as Golgi membranes to the ER in a reversible manner (Doms et al., 1989; Fujiwara et al., 1988; Lippincott-Schwartz et al., 1989 , 1991a Strous et al., 1991) . BHK-21 cells were treated for 90 min with 2 #g/ml of BFA at 37~ to ensure complete translocation of man II to the ER. To follow the movement of man II out of the ER, BFA was removed with several washes of preconditioned medium (20~ in the presence or absence of 10 mM caffeine. Thereafter, the cells were chased for 60, 120, and 180 min at 20~ with (Fig. 3 , d, f, and h, respectively) or without (Fig. 3 , c, e, and g, respectively) caffeine, and then fixed and prepared for immunofluorescence. In Fig. 3 a, the distribution of man II is shown before any drug treatments. In cells treated for 90 min with BFA at 370C, man Figure 2 . The exit of SFV membrane glycoproteins E1 and E2 from the ER in the presence of 10 mM caffeine at different temperatures. BHK-21 ceils infected with SFV ts-1 were incubated for 120 min in the presence of 10 mM caffeine and 50 #g/ml of cycloheximide at 20 (b), 21 (c), 22 (d), 23 (e), 24 (f), 25 (g), and 28~ (h), fixed and processed for immunofluorescence, a shows the distribution of glycoproteins at the restrictive temperature 38.5~ At temperatures 20, 21, 22, and 23~ (b, c, d, and e, respectively), SFV glycoproteins can be found in the ER. At 240C (f), a few cells possess also vesicular labeling ~ arrow). At 25~ in more than half of the cells, SFV glycoproteins can exit the ER and accumulate to perinuclear structures (g). At 28~ virtually all cells show perinuclear localization of El (h). Bar, 20/~m. II is translocated efficiently (in 99 % of the cells) to the ER (Fig. 3 b) . As the cells were incubated for 60 min in a caffeine-free medium at 20~ (Fig. 3 c) , man II appears in peripheral and more central elements (in 47 % of the cells), and also in the ER. After a 120-min chase in caffeine-free medium at 20~ the ER was efficiently emptied and man II was found in a few peripheral elements, but was mainly perinuclearly located (Fig. 3 e) (in 95 % of the cells). After 180 min in caffeine-free medium, man II was found perinuclearly located with no detectable labeling in the ER (Fig. 3 g) (in 98 % of the cells). When the cells were chased after the removal of the BFA in the presence of 10 mM caffeine at 20~ for 60 min, man II failed to exit the ER (Fig. 3 d) (in 98% of the cells). After a 120- (Fig. 3 f ) or 180-rain (Fig. 3 h) chase in the presence of caffeine, man II was still located in the ER (in 94 and 96 % of the ceils, respectively). These results show that 10 mM caffeine and 20~ inhibited efficiently the movement of man II, a resident Golgi membrane protein, out from the ER. This may further suggest that caffeine at reduced temperature inhibits the recovery of the Golgi from the BFA treatment and thus inhibits the membrane traffic from the ER to th