We have re-examined the effects of the ionophore monensin on the Golgi apparatus of sycamore maple suspension-cultured cells using a combination of high pressure freezing, immunocytochemical and biochemical techniques. Exposure of the cells to 10 microM monensin, which reduces protein secretion by approximately 90%, resulted first in the swelling of the trans-Golgi network, then of the trans-most trans-cisterna, the remaining trans-cisternae, and finally of the cis and medial cisternae. We postulate that these different rates of swelling reflect an underlying hierarchy of compartmental acidification with the trans-Golgi network being the most acidic compartment. Recovery occurred in the reverse sequence. Previous studies have suggested that the large swollen vesicles that accumulate in the cytoplasm of monensin-treated cells arise from the swelling and detachment of entire trans-cisternae. However, based on the many membrane blebbing configurations seen in association with the trans-Golgi network and the trans-Golgi cisternae of monensin-treated cells, and the fact that the surface area of the trans-Golgi cisternae is about five times greater than the surface area of the swollen vesicles, it appears that the swollen vesicles are produced by a budding mechanism. After 35–40 min of monensin treatment, cells with smaller, non-swollen, compact Golgi stacks began to appear and rapidly increased in number, contributing > 60% of the cell population after 60 min and > 80% after 100 min. In contrast, large numbers of swollen vesicles persisted in the cytoplasm of all cells for over 100 min. Since azide treatment of monensin-treated cells can prematurely induce the unswelling response and cellular ATP levels drop substantially after 45 min of monensin treatment, we propose that un-swelling of the Golgi stacks is due to a monensin-induced decline in ATP levels in the cells. Immunocytochemical labeling of the high pressure frozen cells with anti-xyloglucan antibodies demonstrated that the concentration of xyloglucan, a hemicellulose, in the swollen vesicles increased with time. This increase in vesicle contents may explain why these swollen vesicles do not contract in parallel with the Golgi stacks. In vivo labeling experiments with [3H]fucose, [3H]UDP-glucose and [3H]leucine demonstrated that monensin-treatment not only inhibited protein secretion, but also cellulose synthesis. Protein synthesis, on the other hand, was reduced only slightly during the first 30 min of treatment, but quite strongly between 30 and 60 min, consistent with the observed drop in ATP levels after > 40 min of exposure to monensin.(ABSTRACT TRUNCATED AT 400 WORDS)
A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex
