Arabinogalactan proteins (AGPs) are extracellular proteoglycans with many O-linked glycan chains. Precursors to many AGPs contain a C-terminal signal for the addition of a GPI-anchor, yet the role of this modification has not been elucidated. NtAGP1, a tobacco precursor to AGP, comprises a signal peptide, an AGP-coding region and a GPI-anchoring signal, and classified as a member of classical AGP. Using green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags and tobacco BY-2 cells as the host, we analyzed the transport and modification of NtAGP1. The fusion protein of GFP or SPO and NtAGP1 expressed in BY-2 cells migrated as a large smear on SDS-polyacrylamide gel. Confocal microscopic analysis indicated that the GFP and NtAGP1 fusion protein localized to the plasma membrane (PM), and fractionation studies of microsomes indicated that the majority of the fusion protein of SPO and NtAGP1 (SPO-AGP) localized to the PM. In contrast, the expression of mutants without a GPI-anchoring signal yielded several forms, and the largest forms migrating as large smears on the gel were secreted into the culture medium, whereas other forms were recovered in the endomembrane organelles. Comparison of the glycan structures of the SPO-AGP recovered in microsomes and the secreted mutant SPO-AGP without a GPI-anchoring signal using antibodies against AGP glycan epitopes indicated that the glycan structures of these proteins are different. These observations indicated that a GPI-anchoring signal is required for both the proper transport and glycosylation of the AGP precursor.

Many precursors of plant arabinogalctan proteins (AGPs) contains 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.