HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Significance The potential of protein therapeutics is limited because most proteins cannot reach the cytosol. Like many cell-penetrating peptides (CPPs), cell-permeant miniature proteins (CPMPs) that embody a penta-Arg motif are endocytosed; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we report that the trafficking of CPMPs into the cytosol requires the homotypic fusion and protein-sorting (HOPS) complex, a membrane-tethering complex that fuses Rab7 + endosomes. CPMPs neither inhibit nor activate HOPS function; instead, HOPS allows CPMPs to traffic into Lamp1 + endosomes as a prerequisite for endosomal escape. The identification of Lamp1 + late endosomes and lysosomes as portals for passing proteins into the cytosol will aid the development of next-generation biologics that overcome the limitations imposed by cellular membranes.