Abstract: BTN2 gene expression in yeast is up-regulated in response to the deletion of BTN1, which encodes the ortholog of a human Batten Disease protein. We isolated Btn2 as a Snc1 v-SNARE binding protein using the two-hybrid assay and examined its role in intracellular protein trafficking. We show that Btn2 is an ortholog of Drosophila and mammalian Hook1 that interacts with SNAREs, cargo proteins, and coat components involved in endosome-Golgi protein sorting. By immunoprecipitation, Btn2 bound the yeast endocytic SNARE complex (e.g. Snc1/2, Tlg1, Tlg2, Vti1), the Snx4 sorting nexin, and retromer (e.g. Vps26, Vps35). In in vitro binding assays, recombinant His6-tagged Btn2 bound GST-Snc1 and GST-Vps26. Btn2-GFP and Btn2-RFP co-localize with Tlg2, Snx4, and Vps27 to a compartment adjacent to the vacuole that corresponds to a late endosome. The deletion of BTN2 blocks Yif1 retrieval back to the Golgi, while the localization of Ste2, Fur4, Snc1, Vps10, CPY, CPS, Sed5, and Sec7 is unaltered in btn2Delta cells. Yif1 delivery to the vacuole was observed in other late endosome-Golgi trafficking mutants, including ypt6Delta, snx4Delta, and vps26Delta cells. Thus, Btn2 facilitates specific protein retrieval from a late endosome to the Golgi, a process which may be adversely affected in Batten Disease patients.