Proximity-dependent biotin identification (BioID) screening identified novel layilin-proximal proteins

Layilin, a type-Ⅰ transmembrane protein, was initially reported as a hyaluronic acid receptor and has since been implicated in cell motility, mitochondrial regulation, immune regulation, and ciliogenesis. However, there remains limited information on its interacting proteins essential for understanding the mechanisms behind these roles. This study aimed to comprehensively identify layilin-interacting proteins using proximity-dependent biotin identification (BioID) screening.

A fusion protein comprising BioID2 of a biotin ligase and the intracellular domain of layilin (BioID2-layilin^Cyt) or BioID2 alone as a control was produced in HEK293 Tet-On cells. In the cells, proteins proximal to the layilin^Cyt were biotinylated by the fused BioID2. Total cellular proteins were extracted and digested by trypsin. Biotinylated peptides were purified and analyzed by LC-MS/MS. Proteins containing biotinylated peptide sequences were identified and subjected to Gene Ontology (GO) enrichment analysis.

Overall, 4594 and 1050 peptides were detected in the BioID2 and BioID2-layilin^Cyt groups, respectively. Among these, 2577 in the BioID2 group and 562 in the BioID2-layilin^Cyt group were biotinylated. Based on these biotinylated peptides, 721 and 173 proteins were identified in the BioID2 and BioID2-layilin^Cyt groups, respectively. Out of the 173 proteins, 19 were exclusively identified in the BioID2-layilin^Cyt group. Excluding the bait protein, 18 layilin-proximal proteins were identified. GO enrichment analysis revealed that they were enriched in biological processes related to protein folding and localization.

Our BioID screening identified putative layilin interactors, providing new insights into the layilin intracellular network. This information lays the groundwork for future investigations into the function of layilin.