FIGURE 6: Effect of NSP1 SPI at the HOPS complex. (A) Uptake of FM4-64 dye to the vacuolar membrane. BY4741 cells were grown in medium containing 4 nM FM4-64 and images taken every 10-20 minutes. The progression of the dye to the vacuolar membrane is clear within 60 minutes. The scale bar is 10 µm. (B) HOPS and CORVET complexes are illustrated based on electron microscopy 3D reconstruction [28] and functional domains [68]. Vps33 and Vps41 were identified as SPIs in the NSP1 screen and are highlighted in purple accordingly. (C) Following a 90-minute induction of GBP or NSP1-GBP expression in a Vps33-GFP strain, FM4-64 was added, and vacuolar staining of cells was quantified at 10, 25, 45 and 60 minutes from dye addition. Error bars indicate 95% binomial confidence intervals. (D) The experiment is as in panel C, but using a Vps8-GFP strain. (E) The data is as in panel C, but with a strain that lacks any GFP, BY4741.

28. Bröcker C, Kuhlee A, Gatsogiannis C, kleine Balderhaar HJ, Hönscher C, Engelbrecht-Vandré S, Ungermann C, and Raunser S (2012). Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Proc Natl Acad Sci 109(6): 1991 LP – 1996. 10.1073/pnas.1117797109

68. Balderhaar HJ kleine, and Ungermann C (2013). CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. J Cell Sci 126(6): 1307–1316. 10.1242/jcs.107805