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

To study the reversibility of the caffeine-induced translocation of p58, BHK-21 cells were treated first with 10 mM caffeine at 20~ for 60 min, followed by several washes with noncaffeine medium. After incubation for an additional 60 min in the absence of caffeine at 20~ the localization of p58 was compared with that at normal temperature (37~ and to that at 20~ without caffeine. In addition, the localization of p58 was compared with that of man II in the same cells. In untreated cells, p58 is located perinuclearly with some labeling in small peripheral vesicles and ER as reported earlier (Saraste and Svensson, 1991) (Fig. 6, no drug) . In cells treated with caffeine at 20~ for 60 min (Fig. 6, 20~ caffeine), the bulk of p58 becomes distributed throughout the cytoplasm while man II still remains perinuclearly located. On the contrary, in control cells without caffeine at 20~ p58 was concentrated to the Golgi area and colocalized with man II (Fig. 6, 20~ control) . The normal localization of p58 was restored when the cells maintained for 60 min in the presence of 10 mM caffeine at 20~ were further incubated for 60 min at 20~ in a caffeine-free medium (Fig. 6, reversion) . The observed reversible translocation of preand cis-Golgi marker protein to the periphery of the cell and the unaltered distribution of a Golgi stack marker protein suggest that caffeine at reduced temperature can be used to manipulate pre-and cis-Golgi elements without affecting the localization of Golgi stack membranes.