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

Since the acquisition of Endo H resistance indicates only that E1 has reached the Golgi stack, we next studied whether SFV glycoproteins can exit the ER at temperatures between 200C and 25~ and whether they are observed to accumulate in intermediate elements between the ER and the Golgi compartment. BHK-21 cells were infected with SFV ts-1 for 1 h, shifted to restrictive temperature (38.5~ and grown there for 4 h. The cells were then shifted to 20, 21, 22, 23, 24, 25, and 280C, and chased for 2 h in the presence or absence of 10 mM caffeine. To be able to follow the movement of presynthetized E1 and E2, the chase mediums contained 50 t~g/ml of cycloheximide. In cells incubated at 20, 21, 22, and 23~ in the presence of caffeine, the SFV glycoproteins remained in a reticular structure ( Fig. 2 , b, c, d, and e, respectively) identical to the ER labeling observed in the control cells fixed at the restrictive temperature (38.5~ (Fig. 2 a) . At 24~ only a few cells showed perinuclear vesicular labeling (Fig. 2 f, arrow) in addition to ER labeling. At 25~ the number of cells displaying a perinuclear pattern of labeling increased significantly compared with the situation at 24~ (Fig. 2 g) . As was previously reported , the SFV glycoproteins accumulated in perinuclear structures at 28~ in the presence of caffeine (Fig. 2 h) . In control cells, the glycoproteins were transported to the trans-Golgi region already at 200C as described earlier (Marlin and Simons, 1983; Saraste and Kuismanen, 1984) , and at higher temperatures increased labeling of the plasma membrane was observed (data not shown).