Structural determinants and biochemical characterization of LORELEI as a GPI-anchored protein

Glycosylphosphatidylinositol (GPI)-anchored proteins play critical roles in plant development, reproduction, and environmental responses. However, their biochemical characterization in plants remains limited. LORELEI, a putative GPI-anchored protein involved in pollen tube reception and early seed development in Arabidopsis (Arabidopsis thaliana), has lacked direct biochemical evidence confirming its GPI anchoring. This study employed biochemical approaches to validate LORELEI as a GPI-anchored protein. We demonstrate that ectopic expression of wild-type LORELEI fused to a reporter protein in vegetative tissues associates with a detergent-resistant membrane fraction and is sensitive to GPI-specific cleavage enzymes, thereby confirming that it is a GPI-anchored protein. Moreover, we show that mutations in the ω-sites or the GPI attachment signal of LORELEI disrupt its membrane localization, highlighting the necessity of these structural elements for proper GPI anchoring. Analysis of a reporter-fused LORELEI protein in the loss-of-function mutant of GPI8, which encodes the catalytic subunit of the GPI transamidase complex involved in GPI anchor addition, results in the appearance of prominent higher molecular weight bands, further supporting its role in GPI anchoring of the LORELEI protein. This study provides direct biochemical evidence confirming LORELEI as a GPI-anchored protein and sheds light on the structural determinants required for its GPI anchoring. Additionally, our study demonstrates that heterologous ectopic expression of LORELEI in vegetative tissues provides a viable alternative for the biochemical characterization of GPI-anchored proteins predominantly expressed in hard-to-access and small-sized female gametophytes. Our findings underscore the role of GPI anchoring in membrane localization and biosynthesis of GPI-anchored proteins in plants.