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

We have previously shown that at 20~ 10 mM caffeine blocks efficiently the movement of newly synthesized SFV glycoproteins out from the ER . Normally, in the absence of caffeine at 20~ protein transport occurs to the level of the tmns-Golgi (Matlin and Simons, 1983; Saraste and Kuismanen, 1984) . The exact Figure 1 . Determination of the temperature threshold allowing SFV E1 to reach the Golgi in the presence (a) or absence (b) of 10 mM caffeine. BHK-21 cells were infected with SFV ts-1 temperature-sensitive mutant, labeled with [35S]methionine and chased in the presence or absence of 10 mM caffeine at 20, 21, 22, 23, 24, 25, 26, and 28~ for 3 h. The cells were then solubilized, immunoprecipitated with monospecific polyclonal antibodies to SFV El, treated with Endo H, and run in a 10% SDS-PAGE under reducing conditions. From a it can be observed that SFV E1 remains Endo H sensitive in the presence of 10 mM caffeine up to 240C. E1 becomes gradually resistant to Endo H treatment at 250C and at higher temperatures, indicating the arrival of E1 to the Golgi stack. In b the sensitivity of E1 to Endo H treatment in the absence of caffeine at different temperatures is shown. In the absence of caffeine, E1 becomes Endo H resistant already at 20~ (b). P, pulse. temperature threshold that allows SFV E1 membrane glycoprotein to reach the Golgi stack in the presence of caffeine was analyzed by following the maturation of SFV El-linked glycans to the Endo H-resistant form. We also carried out immunofluorescence experiments to analyze the exact temperature that allows SFV glycoproteins to exit the ER. These experiments together should further reveal whether caffeine at reduced temperature could arrest the ER-to-Golgi traffic in possible intermediate steps.