Secreted arabinogalactan protein from salt-adapted tobacco BY-2 cells appears to be glycosylphosphatidyl inositol-anchored and associated with lipophilic moieties.

Arabinogalactan proteins (AGPs) are plant extracellular proteoglycans associated with the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. This moiety is thought to be cleaved by phospholipase for secretion. Salt-adapted tobacco BY-2 cells were reported to secrete large amounts of AGPs into the medium. To investigate this mechanism, we expressed a fusion protein of tobacco sweet potato sporamin and AGP (SPO-AGP) in BY-2 cells and analyzed its fate after salt-adapting the cells. A two-phase separation analysis using Triton X-114 indicated that a significant proportion of SPO-AGP in the medium was recovered in the detergent phase, suggesting that this protein is GPI-anchored. Differential ultracentrifugation and a gradient density fractionation implicated extracellular vesicles or particles with SPO-AGP in the medium. Endogenous AGP secreted from salt-adapted and nontransgenic BY-2 cells behaved similarly to SPO-AGP. These results suggest that a part of the secreted AGPs from salt-adapted tobacco BY-2 cells are GPI-anchored and associated with particles or vesicles.

Glycosylphosphatidylinositol-anchoring is required for the proper transport and extensive glycosylation of a classical arabinogalactan protein precursor in tobacco BY-2 cells.

Many precursors of plant arabinogalactan proteins (AGPs) contain a C-terminal glycosylphosphatidylinositol (GPI)-anchoring signal. Using NtAGP1, a classical tobacco AGP, as a model, and green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags, we analyzed the localization and modification of AGP and its mutant without GPI-anchoring signal (AGPΔC) in tobacco BY-2 cells. The NtAGP1 fusion proteins migrated as large smear on SDS-polyacrylamide gel, and these proteins also localized preferentially to the plasma membrane. In contrast, fusions of AGPΔC with GFP and SPO yielded several forms: The largest were secreted, whereas others were recovered in the endomembrane organelles, including vacuoles. Comparison of the glycan structures of the microsomal SPO-AGP and the secreted SPO-AGPΔC using antibodies against the glycan epitopes of AGP indicated that the glycan structures of these proteins are different. These observations indicate that GPI-anchoring is required for the proper transport and glycosylation of the AGP precursor.