Selective translocation of different markers in the ante- and retrograde pathways between the Golgi apparatus and the rough endoplasmic reticulum in a hybridoma cell line

Abstract

We examined the effects of brefeldin A (BFA, 10 micrograms/ml), an inhibitor of protein transport, on the redistribution of different markers of the Golgi apparatus (GA) in hybridoma H35 cells to examine selective transport of marker molecules between the rough endoplasmic reticulum (RER) and the GA. In H35 cells, the GAs had several cisternae with cis and trans faces as deduced by morphology such as relationship with RER and secretory granules. Thiamin pyrophosphatase (TPPase) was distributed in the trans elements, mannosidase II (man II) was in the cis-medial elements, and deposits of Zinc-Iodide-Osmium (ZIO) staining were localized in the cis/intermediate compartment. Upon BFA treatment for 5 min, man II and TPPase were redistributed in all cisternae. After 10 min of BFA treatment, TPPase activity was observed only in the RER, while the cis/intermediate compartment as evidenced by ZIO staining and man II remained. Upon clearance of BFA from the medium, cisternal structures with man II and ZIO staining reappeared at 30 min. TPPase activity was detected in the GA only after 120 min. Thus, in the retrograde pathway, the trans marker, TPPase moves earlier than the cismedial markers, man II and ZIO staining, whereas in the antegrade pathway, the cis-medial markers move earlier than the trans marker. These results suggest that BFA first alters the characteristic enzyme localization before the GA vanishes into the RER, and that selective transport mechanisms may exist for components of different stacks of the GA.