Fig. 3. Osh1p localizes and purifies with Nvj1p at NV junctions and vacuole-independent rafts. (A) Osh1p requires Nvj1p in order to localize to NV junctions and vacuole-independent rafts. nvj1 (i,ii) or vac8 (iii,iv) cells expressing GFP-Osh1p and harboring either P_CUP1-NVJ1-Fc or empty vector were analysed by confocal microscopy at log phase. Expression of Nvj1p-Fc or empty vector was induced for 1.5 hours with CuSO₄. Vacuolar membranes (red) and nuclear chromatin (blue) were stained with FM4-64 and Hoechst, respectively, and overlayed with GFP-Osh1p. The localization of GFP-Osh1p to NV junctions appears yellow in the overlay (ii, arrow). In vac8 cells, GFP-Osh1p localizes to non-yellow vacuole-independent patches on the nuclear surface (iv, arrow). (B) GFP-Osh1p specifically purifies with Nvj1p-Fc in nvj1 and vac8 cells. Extracts of nvj1 and vac8 cells expressing GFP-Osh1p and harboring either P_CUP1-Nvj1p-Fc or empty vector were prepared as described in Materials and Methods. Nvj1p-Fc was purified from whole-cell lysate using protein-A-conjugated agarose pre-absorbed with 1% bovine serum albumin. The co-purification of GFP-Osh1p with Nvj1p-Fc was assayed by immunoblot using polyclonal anti-GFP antibodies. (C) Examples of vacuole-independent rafts of Nvj1p-EYFP in vac8 cells after induction with CuSO₄. These vacuole-independent patches occur on the nuclear surface as revealed by Hoechst (blue) but fail to localize with vacuolar membranes (red).