PROTEIN SEGREGATION IN PERIPHERAL 15-DEGREES-C INTERMEDIATES IN RESPONSE TO CAFFEINE TREATMENT

Previous studies have shown that caffeine treatment at 20 degrees C ca uses the intermediate compartment protein p58 to redistribute from the Golgi region without affecting the localization of the Golgi stack pr otein mannosidase II (J. Jantti, E. Kuismanen, J. Cell Biol, 120, 1321 -1335 (1993)). Here we have dissected further the effect of caffeine o n transport of Golgi and intermediate compartment proteins from the ce ll periphery to the perinuclear Golgi region, To accumulate proteins i n the peripheral membranes, BHK-21 cells were treated with brefeldin A to redistribute marker proteins towards the ER, Following BFA wash-ou t and subsequent incubation at 15 degrees C, p58, the coat protein bet a-COP, and Man II were all localized in the peripheral 15 degrees C-in termediates. When the cells were shifted from 15 degrees C to 20 degre es C all the proteins were recentralized to the Golgi region, However, if the temperature shift was carried out in the presence of 10 mM caf feine, p58 and P-COP maintained their peripheral localization, whereas Man II was transported to the Golgi region, The results indicate that caffeine at 20 degrees C does not block the centralization of Man II from peripheral sites to the central Golgi region, Therefore, its effe ct on ER to Golgi transport appears to be manifested specifically at E R exit, Furthermore, our results indicate that segregation of intermed iate compartment and Golgi stack proteins can occur at the level of th e peripheral 15 degrees C-intermediates. Immunoelectron microscopic lo calization of p58 and Man II showed that these peripheral intermediate s consisted of tubules and small stacks of cisternae, Within the tubul ar intermediates both p58 and Man II appeared to segregate to membrane subdomains, Finally, examination of serial and thick sections support the idea that the stacked structures can be generated from tubular in termediates.