Fig. 4.

Two Rab-binding sites on a single HOPS complex. (A) Interaction of Ypt7 with Vps39. Rab pull-down with GTPγS- or GDP-loaded Rabs Ypt7, Vps21, and Ypt7 was done with purified HOPS or the HOPS subcomplexes. Bound proteins were eluted and analyzed on SDS/PAGE, followed by Western blotting against the CbP-tag (indicated with *). A representative Coomassie gel shows GST-Rabs present in the pull-down. (B) HOPS and Ypt7-His₆ were incubated, cross-linked, and separated on a glycerol gradient. The complexes were then labeled with Ni-NTA-nano-gold and imaged in the electron microscope. Left: Raw particle images. Right: Localization of the gold-label graphically mapped to the model of HOPS as shown in Fig 3. (Scale bar, 10 nm.) (C) Localization of GFP-tagged HOPS subunits. All HOPS subunits were genomically tagged at their C terminus with GFP and localized by fluorescence microscopy in wild-type (wt) and ypt7Δ cells. (Scale bar, 10 μm.) (D) Model of HOPS function in fusion. SNAREs are in red. Details in text.