The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.Secretion of proteins from animal cells is a process which, while not fully understood, has been well characterized. Secretion is known to involve the entrance of the protein to the endomembrane system (which requires the presence of a signal sequence on the protein), movement of the protein through the ER and the Golgi body, and fusion of secretory vesicles with the plasma membrane (20,22
ABSTRACrCarbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth (8,9).Coupled with the use ofsuspension cultures, carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy can provide an extremely powerful technique for characterizing metabolic reactions (2, 4, 13). Unlike carbon-14 studies which simply allow the determination of the extent of incorporation of a radiolabel into a given metabolite (after often extensive isolation, separation, and chromatographic purification of the metabolite), the use of specifically '3C-labeled substrates allows the fate of a specific carbon atom to be followed through many enzymic transformations of the original substrate. Not only the distribution of the label in the various intermediary metabolite pools of the cell but perhaps most importantly, the specific location ofthe labeled carbon atom in the various intermediate's molecular structure can be determined (15,16). This information can be obtained ' Present
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