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

Recent work by many laboratories has shown that the transport between the ER and the Golgi is mediated by transport vesicles. These transport vesicles appear to exit throughout the ER and to concentrate perinuclearly along the microtubules in vivo (Saraste and Svensson, 1991) . Only a few markers for these intermediate elements are known. Two membrane proteins, p58 (Saraste et al., 1987) and p53 (Schweizer et al., 1988) , are localized to the intermediate elements and the cis-Golgi. In addition, the KDEL receptor, responsible for the retention of soluble ER proteins (Pelham, 1988; Pelham, 1989) , is likely to be located at least in some degree to these membranes . There is accumulating knowledge on the com- in the Golgi area in cells treated with caffeine at 20~ BHK-21 ceils were incubated at 20~ with (a) or without (b) 10 mM caffeine, fixed, and processed for immunoperoxidase electron microscopy with antibodies to p5& The alteration of the Golgi morphology in caffeine-treated cells (a) can be observed. Arrows mark the p58-positive structures found in the Golgi region both in caffeine-treated (a) and in control cells (b). In caffeine-treated cells (a) the amount of p58-1abeled membranes seems to be lower compared with that observed in control cells (b). Bar, 500 nm. Figure 9 . Effect of caffeine at 20~ on the localization of/3-COP in BHK-21 cells. BHK-21 cells were grown for 60 min in the presence (c) or absence (b) of 10 mM caffeine at 20~ fixed, and processed for immunofluorescence with antibodies to fl-COP, a shows the normal distribution of fl-COP in BHK-21 cells. Caffeine at reduced temperature does not seem to affect the distribution of fl-COP (c) compared with that in control cells incubated only at 20~ (b) or to that observed in cells without any treatment (a). Bar, 20 #m. plexity of the molecular machinery involved in the ER-to-Golgi transport (for review see Rothman and Orci, 1992) , based on work with yeast mutants (Novick et al., 1980) and cell-free transport assays (Fries and Rothman, 1980; Balch et al., 1984) . In studies concerning the organization of the anterograde and the retrograde membrane traffic, the reversible effect of BFA on the organization of the Golgi complex has provided important new information (Pelham, 1991; Klausner et al., 1992) . Our previous report and the results in the present study suggest that caffeine is a new potential tool to dissect steps in the exocytic pathway.