Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspen sion-cultured BY-2 tobacco cells simultaneously with another recombinant va cuolar protein, the sweet potato sporamin. In contrast to previous observat ions in different transgenic plant systems when expressed in BY-2 tobacco c ells, phytohemagglutinin is mostly but not exclusively targeted to the vacu ole. Indeed, a small amount of recombinant phytohemagglutinin is secreted i nto the culture medium of tobacco cells. Furthermore part of this extracell ular phytohemagglutinin has no lectin activity and presents an abnormal gly cosylation consistent with higher accessibility of glycans N-linked to thes e extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occu rs regardless of recombinant protein expression level. Consequently, missor ting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 c ells with drugs, such as monensin and wortmannin, increases even more the t ransport of phytohemagglutinin to the cell surface through a general inhibi tion of the sorting mechanisms of vacuolar proteins. The sensitivity to wor tmannin is similar for the sorting of phytohemagglutinin and endogenous tob acco chitinase and beta-1,3-glucanase, suggesting that phytohemagglutinin a nd COOH-terminal propeptide mediated vacuolar sorting share similar mechani sms. A characterization of glycans N-linked to extracellular phytohemagglut inin secreted by monensin- or wortmannin-treated transgenic tobacco cells i llustrates that in contrast with monensin, wortmannin completely inhibits t he sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes. (C) 1999 Editions scientifiques et medicales Elsevier SAS.