The fusion (right) of early endosomes (left) is inhibited by Hrs.

Endocytosed proteins come together in the early endosome to be sorted to different locations. Some endosome fusion is needed to promote this mixing, but the process must be regulated both to stop the formation of one giant early endosome and to promote eventual sorting. Now, Sun et al. (page 125) have developed a clever assay for studying homotypic endosome fusion and used it to uncover a surprising new fusion inhibitor activity for an early endosome protein called Hrs.The authors allowed different populations of HeLa cells to endocytose EGF linked to one of two different fluorophores, and then isolated pools of endosomes or lysosomes from the cells. When the isolated organelles were allowed to fuse in cell-free reactions, the mixing of their contents caused fluorescence resonance energy transfer between the two fluorophores, providing an easily quantified readout.

Using this technique, Sun et al. show that Hrs, a mammalian protein found primarily on early endosomes, specifically inhibits homotypic fusion of early endosomes, and that the coiled-coil domain of Hrs is necessary and sufficient for this activity. The Hrs coiled-coil domain binds to a SNARE complex consisting of SNAP-25 and syntaxin 13, thus preventing the binding of VAMP2, which is required for fusion.

The results are surprising: first, because Hrs was thought to bind to endosomes through a phosphorylated lipid rather than a specific protein receptor; and second, because SNAP-25 was thought to be involved only in exocytosis, whereas the new work shows a requirement for SNAP-25 in an endocytic pathway.

Sun et al. suggest that although the Hrs coiled-coil domain prevents fusion, other domains of Hrs might simultaneously direct cargo sorting or endosome movement. The combined activities of preventing an early endosome from fusing with its neighbors and moving it toward its destination would help Hrs direct the sorting and separation of endosomal cargo. The authors are now examining the regulation of the Hrs-containing SNARE complex and developing an automated, high-throughput version of their assay for future studies. ▪