Bidirectional transfer of Engrailed homeoprotein across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates to the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer in HeLa cell culture, we demonstrate a core role of the phosphatidylinositol-4,5-biphosphate (PIP2) together with cholesterol in the translocation of Engrailed2 (EN2) homeoprotein across the plasma membrane. Using drug and enzymatic treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement of PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased with a paracrine-deficient mutant of EN2. We propose that the bi-directional plasma membrane translocation events occurring during homeoprotein secretion and internalization respectively are parts of a common process.