Summary Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in tumor-induced angiogenesis. FGF2 is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here we demonstrate that both PI(4,5)P 2 -dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further reveal cholesterol to enhance FGF2 binding to PI(4,5)P 2 -containing lipid bilayers in a fully reconstituted system. Based on extensive atomistic molecular dynamics simulations and membrane tension experiments, we propose cholesterol to modulate FGF2 binding to PI(4,5)P 2 by (i) increasing head group visibility of PI(4,5)P 2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P 2 molecules triggering FGF2 oligomerization and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 towards the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Cholesterol promotes both head group visibility and clustering of PI(4,5)P2driving unconventional secretion of Fibroblast Growth Factor 2