HOPS - Molecular Insights into Vesicle Sorting

Abstract

For intracellular separation between metabolic and physiological entities eukaryotic cells developed a complex compartmentalization by intracellular membranes. To allow directed transport of cargo and membranes to their destination organelle, they use a dynamic but highly specific vesicular transport system. The recognition of vesicles at their target compartment is initiated by the reversible interaction of so-called tethering complexes and Rab-GTPases (e.g. Ypt7) prior to SNARE-mediated membrane fusion. Although structural data on coiled-coil and Rab-independent tethers exist, molecular insight into the structure of a Rab-binding multisubunit tethering complexes (MTCs), like the endosomal CORVET and the vacuolar HOPS complex, has been lacking to date. Here we analyzed the HOPS complex structure using transmission electron microscopy (TEM) combined with single particle analysis. We show that the heterohexameric HOPS is highly flexible forming a seahorse-like structure. Surprisingly, the two Rab-binding proteins Vps39 and Vps41 are at opposite ends, implicating that HOPS bridges Ypt7-positive membranes. We also specified the parts of the SNARE complex that bind to the Vps33 subunit, which is proximal to the Rab-binding site Vps41. This suggests that HOPS coordinates Rab-mediated tethering with SNARE-driven fusion. Taken together our data demonstrate that the conserved vacuolar/lysosomal HOPS tethering complex combines different activities; the vesicle recognition by Rab-binding, bridging of vesicular and target membrane and the activation of SNARE mediated fusion.