Structural Determinants and Biochemical Characterization of LORELEI as a GPI-Anchored Protein

Abstract Glycosylphosphatidylinositol-anchored proteins play critical roles in plant development, reproduction, and environmental responses. However, their biochemical characterization in plants remains limited. LORELEI, a putative glycosylphosphatidylinositol-anchored protein involved in pollen tube reception and early seed development in Arabidopsis thaliana , has lacked direct biochemical evidence confirming its glycosylphosphatidylinositol anchoring. This study employed biochemical approaches to validate LORELEI as a glycosylphosphatidylinositol-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 glycosylphosphatidylinositol-specific cleavage enzymes, thereby confirming that it is a glycosylphosphatidylinositol-anchored protein. Moreover, we show that mutations in the ω-sites or the glycosylphosphatidylinositol attachment signal of LORELEI disrupt its membrane localization, highlighting the necessity of these structural elements for proper glycosylphosphatidylinositol anchoring. Analysis of a reporter-fused LORELEI protein in the loss of function mutant of glycosylphosphatidylinositol 8 , which encodes the catalytic subunit of glycosylphosphatidylinositol transamidase complex involved in glycosylphosphatidylinositol anchor addition, results in the appearance of prominent higher molecular weight bands, further supporting its role in glycosylphosphatidylinositol anchoring of the LORELEI protein. This study provides direct biochemical evidence confirming LORELEI as a glycosylphosphatidylinositol-anchored protein and sheds light on the structural determinants required for its glycosylphosphatidylinositol anchoring. Additionally, our study demonstrates that heterologous ectopic expression of LORELEI in vegetative tissues provides a viable alternative for the biochemical characterization of glycosylphosphatidylinositol-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.