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Fig. 1. Osh1p is sequestered from cytoplasmic and Golgi pools to NV junctions through an Nvj1p-mediated mechanism. (A) Localization of GFP-Osh1p as a function of NVJ1 expression. Cells expressing plasmid-encoded GFP-Osh1p and harboring P_CUP1-NVJ1 or empty vector were analysed at log phase. P_CUP1-NVJ1 expression was maintained at a basal level (iii,iv) or induced for 1.5 hours with CuSO₄ (v,vi) similar to empty vector control cells (i,ii). Nuclear chromatin (blue) and vacuolar membranes (red) were stained with Hoechst and FM4-64, respectively, and overlayed with GFP-Osh1p (bottom). The fluorescence of GFP-Osh1p increases at yellow-labeled NV junctions (v,vi) at the expense of Golgi and cytoplasmic pools (i,iii). (B) Localization of GFP-Osh1p as a function of mistargeted HA-Nvj1p expression. nvj1 cells expressing GFP-Osh1p and harboring P_CUP1-HA-NVJ1 or empty vector were monitored at log phase. P_CUP1-HA-NVJ1 expression was maintained at a basal level (iii,iv) or induced for 1.5 hours with CuSO₄ (v,vi) similar to empty vector control cells (i,ii). Nuclear chromatin (blue) and vacuolar membranes (red) were stained with Hoechst and FM4-64, respectively, and overlayed with GFP-Osh1p (bottom). (C,D) Immunoblot analysis of GFP-Osh1p levels in whole-cell extract as a function of NVJ1 (C) or HA-NVJ1 (D) expression. Wild-type extracts lacking GFP- or HA-tagged proteins are labeled as N.

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