HOPS-Dependent Endosomal Escape Demands Protein Unfolding

Abstract

The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the translocation of a protein involves two discrete steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules can achieve high protein uptake, but few are capable of efficient endosomal escape. One exception to this rule is ZF5.3, a mini-protein that exploits elements of the natural endosomal maturation machinery to translocate across endosomal membranes. Although some ZF5.3−protein conjugates are delivered efficiently to the cytosol or nucleus, overall delivery efficiency varies widely for different cargoes with no obvious design rules. Here we show that delivery efficiency depends on the ability of the cargo to unfold. Using fluorescence correlation spectroscopy, a single-molecule technique that precisely measures intracytosolic protein concentration, we show that regardless of size and pI, low-T m cargoes of ZF5.3 (including intrinsically disordered domains) bias endosomal escape toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal, even if the T m is high. These findings imply a novel pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.

Requirements for efficient endosomal escape by designed mini-proteins

“…Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells 7 , and guides multiple classes of proteins, including enzymes [8][9][10] , transcription factors 11 , and monobodies 10,12 , into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher 7,10 . The egress of ZF5.3 from endosomes occurs through a biochemically distinct pathway that demands HOPS, a ubiquitous tethering complex that mediates endosomal maturation 13 .…”

“…The median fluorescence intensity (MFI) for the Rhodamine channel was reported for 10,000 gated cells, with at least 2 biological replicates per condition. Confocal microscopy and FCS were performed as previously described 10 . Briefly, all experiments were performed at 37 °C and 5% CO 2 on a STELLARIS 8 microscope (Leica Microsystems) equipped with a HC Plan-Apo 63x/1.4NA water immersion objective (used for FCS measurements).…”

“…ZF5.3 is a compact, rationally designed mini-protein that escapes from the endosomes of multiple cell types with high efficiency 6,7 . Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells 7 , and guides multiple classes of proteins, including enzymes [8][9][10] , transcription factors 11 , and monobodies 10,12 , into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher 7,10 .…”

“…The egress of ZF5.3 from endosomes occurs through a biochemically distinct pathway that demands HOPS, a ubiquitous tethering complex that mediates endosomal maturation 13 . Egress of ZF5.3 and covalent ZF5.3-conjugates from endosomes proceeds without leakage of other intralumenal components 13 , and is especially efficient when the cargo protein is small, intrinsically disordered, or unfolds at a temperature of 35 o C or lower 10 . But other than the requirement for unfoldable cargo 10 and an intact HOPS complex 13 , there is little known about how ZF5.3 traverses the limiting endocytic membrane.…”

“…Egress of ZF5.3 and covalent ZF5.3-conjugates from endosomes proceeds without leakage of other intralumenal components 13 , and is especially efficient when the cargo protein is small, intrinsically disordered, or unfolds at a temperature of 35 o C or lower 10 . But other than the requirement for unfoldable cargo 10 and an intact HOPS complex 13 , there is little known about how ZF5.3 traverses the limiting endocytic membrane. It is especially unclear why the endosomal membrane is favored over, say, the plasma membrane.…”