PIP₂ holds the key for engrailed-2 homeoprotein intercellular trafficking

Homeoproteins are transcription factors that can act in a paracrine manner to regulate development, but they lack a secretion signal to allow their translocation across the plasma membrane. Other soluble proteins such as the basic fibroblast growth factor (FGF2), which also does not encode secretion-peptide signals, rely on unconventional protein secretion (UPS) pathways, but how homeoproteins are secreted is still not well understood. In this study, Alain Joliot and colleagues (Amblard et al., 2020) demonstrate that the chicken homeoprotein engrailed-2 (EN2) binds directly to phosphatidylinositol (4,5)-bisphosphate (PIP₂) for its membrane transfer via a UPS pathway. By combining two different techniques to allow the monitoring of protein secretion in cultured cells, the authors were able to analyse EN2 translocation in response to modulation of PIP₂ levels; a decrease in PIP₂ by dephosphorylation reduced EN2 secretion, whereas expression of phosphatidylinositol 4-phosphate 5-kinase type-1 alpha (PI4P5K) increased PIP₂ levels and, consequently, EN2 secretion. In addition, PIP₂ is involved in EN2 internalisation, suggesting that both routes of EN2 bidirectional trafficking require PIP₂. Finally, two tryptophan residues that are essential for the paracrine function of EN2 appear to be necessary for PIP₂ binding. This study thus identifies the molecular requirements for EN2 intercellular trafficking, as well as the similarities to UPS of other proteins.