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

In the present study, we have dissected the compartmental organization of the ER-to-Golgi interface using the transport inhibition exerted by 10 mM caffeine at 20"C . The temperature threshold inhibiting the exit of SFV glycoproteins from the ER was found to be 23-24~ At 25~ SFV E1 reached the Golgi stack as indicated by the Endo H analysis. Some variation, however, was observed in the temperature dependence between individual cells close to the threshold temperature. Therefore, in other experiments, 20~ in the presence of caffeine was used. To study whether the transport of cellular proteins is also affected, man II was translocated with BFA to the ER, whereafter BFA was washed away and the movement of man II was followed in the absence or presence of 10 mM caffeine at 20~ As was the case with virus proteins, man II was unable to exit from the ER in the presence of 10 mM caffeine at 20~ during a 180-min chase. In contrast, during a 180-min chase in the control cells, ER was efficiently emptied and man II accumulated perinuclearly in the absence of caffeine at 20~ It is therefore suggested that caffeine affects the movement of membranes between the ER and the Golgi complex. It is, however, necessary to follow the movement of other cellular proteins and, for example, lipids between the ER and the Golgi in the presence of caffeine at reduced temperature to obtain a clear picture of this matter. We have also tested that the ER-exit inhibition by caffeine functions in SFV-infected normal rat kidney cells (data not shown), indicating that the observed effect is not specific for one cell type.