Novel plasma membrane proteins in nicotl4na tabacum (gpi-anchored proteins)

Proteins may be associated with the outer leatlet of t he plasma membrane via a glycan linkage to the inositol head group of lipid mlecules. Such g/ycosyl phosphatidylinositol (GPI)-anchored proteins are widt, spread in higher eukaryotes such as yeasts and protozoa. To (late, however, there have been f(,w reports of GPI-anchored proteins in plants. Recent evidence suggests alkaline phosphatase in the aquatic monocot Spivdela oIigorr'hiza and nitrate reductase in the green algae Chlorella saecharophila are (;Pl-anchored proteins. Our study provides the first evidence for GPl-anchored proteins in a higher terrestrial plant. An assay was adapted from mammalian cells to detect GPl-anchored proteins on the surface of protoplasts isolated from .Vicotiana tabacum. The assay employs cell surface labelling with biotin, enzymatic hydrolysis of the GPI-anchor by phosphatidylinositol-specific phospholipase C and temperatureinduced phase separation of proteins. Using this assay six GPI anchored proteins were detected in suspension culture (:ells and seven proteins detected in leaf tissue. We are currently investigating the targeting of proteins to the plant plasma membrane using GPI-anchor attachment signals from yeast and mammalian origin. Constructs have been made containing the reporter gene endoglucanase E' from Clostridium thermocelhtm fused to either plant or mare malian ER-targeting signals. The results of l ransient {,xpression assays in N. tabaeurn cells will be presented.