Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum

Recent studies have demonstrated the existence of glycosyl-phosphatidylinos itol (GPI)-anchored proteins in higher plants. In this study we tested whet her GPI-addition signals from diverse evolutionary sources would function t o link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplas ts were transiently transfected with a truncated form of the Clostridium th ermocellum endoglucanase E reporter gene (celE') fused with a tobacco secre tion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-ter minus. The yeast and plant C-terminal signals were found to be capable of d irecting the addition of a GPI-anchor to the endoglucanase protein (EGE') a s shown by the sensitivity of the lipid component of GPI to phosphatidylino sitol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammali an signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was n ot linked to the EGE' protein indicating the necessity for the missing amin o acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addit ion signal sequences for yeast and plant cells.