Monensin-induced redistribution of enzymes and products from Golgi stacks to swollen vesicles in plant cells

Abstract

We have tested the hypothesis that monensin treatment of plant cells leads to the transfer of Golgi enzymes to the monensin-induced swollen vesicles using sycamore maple suspension-cultured cells and immunolabeling techniques. Cells treated for 20 and 60 min with 10 microM monensin were labeled with anti-V-H(+)-ATPase, anti-xyloglucan (XG) sidechain (CCRC-M1), anti-pectic polysaccharide (JIM7 and CCRC-M2), and anti-N-glycan (anti-beta Xyl, and anti-alpha Fuc) antibodies. Our results demonstrate that monensin causes most H(+)-ATPase to be displaced from the Golgi cisternae (label distribution in control cells: 11% cis, 32% medial, 57% trans cisternae) to the swollen vesicles, which explains why these vesicles remain swollen after detachment from the Golgi/trans Golgi network cisternae. We also show that the content of complete XG molecules (defined by completed trisaccharide sidechains) in the swollen vesicles increases 1.5-fold between the 20 and 60 min samples, suggestive of the transfer of functional XG backbone and sidechain synthesizing enzymes from the trans Golgi compartment to the swollen vesicles. In contrast, no increase in either anti-pectin antibody or N-glycan antibody labeling of the swollen vesicles was observed between the 20 min and 60 min monensin samples. Both of these latter types of molecules depend for their synthesis on enzymes located in multiple membrane compartments upstream from the trans Golgi cisternae, which greatly decreases the probability of transfer of complete enzyme systems to the swollen vesicles. Thus these latter findings do not contradict the anti-H(+)-ATPase and the anti-XG labeling data, which strongly support the Golgi enzyme displacement theory.