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Fig. 4. Osh1p accumulates in PMN structures dependent on NVJ1 but is not required for NV-junction formation or PMN. (A,B) Localization of GFP-Osh1p to PMN structures in wild-type (A) and atg7 (B) cells upon nitrogen starvation. Cells were grown to high OD₆₀₀ (1.0), shifted to SD-N medium and analysed by confocal microscopy after 3 hours of starvation. Vacuolar membranes (red) and nuclear chromatin (blue) were stained with FM4-64 and Hoechst, respectively. GFP-Osh1p localizes to yellow-labeled PMN structures in the overlay. (C) GFP-Osh1p increasingly accumulates within PMN structures proportional to the expression of NVJ1. Cells expressing GFP-Osh1p and harboring P_CUP1-NVJ1 (ii) or empty vector (i) were grown to low OD₆₀₀ (0.3), shifted to SD-N medium and analysed by confocal microscopy after 3 hours of starvation. Vacuolar membranes (red) and nuclear chromatin (blue) were stained with FM4-64 and Hoechst, and overlayed with GFP-Osh1p. PMN structures (arrows) appear yellow in the overlay and increase in size relative to NVJ1 expression. (D) Localization of Nvj1p-EYFP in osh1 cells before and after nitrogen starvation. Log-phase osh1 cells harboring P_GAL1-NVJ1-EYFP were grown briefly in galactose to induce Nvj1p-EYFP and analysed in synthetic complete (SC) or nitrogen starvation medium (SD-N) after glucose repression. Nuclear chromatin and vacuolar membranes were stained with Hoechst and FM4-64, respectively. Arrows indicate PMN structures.

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