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First published online 14 September 2004
doi: 10.1242/jcs.01372

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Nvj1p is the outer-nuclear-membrane receptor for oxysterol-binding protein homolog Osh1p in Saccharomyces cerevisiae

Department of Biology, University of Rochester, 436 Hutchison Hall, Rochester, New York, NY 14627, USA

View larger version (53K): [in a new window]   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 PCUP1-NVJ1 or empty vector were analysed at log phase. PCUP1-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 PCUP1-HA-NVJ1 or empty vector were monitored at log phase. PCUP1-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.

View larger version (109K): [in a new window]   Fig. 2. Nvj1p localizes to the outer leaflet of the nuclear membrane at NV junctions. Immunogold localization of GFP-Nvj1p (A) and Nvj1p-GFP (B) by electron microscopy. Both GFP-tagged Nvj1p reporters were induced in wild-type cells for 1 hour with CuSO4 before cryofixation and immuno-EM analysis. (A) GFP-Nvj1p, a nonfunctional reporter, is mislocalized from NV junctions. Colloidal-gold-conjugated antibodies directed against GFP-Nvj1p localize to the surface of cytoplasmic vacuoles away from the nuclear membrane. Nucleus denoted as N, vacuoles labeled as V. (B) Nvj1p-GFP, a functional reporter, localizes specifically to NV junctions. Colloidal-gold-conjugated antibodies directed against Nvj1p-GFP label the surface of nuclei at NV junctions (i). Higher magnification reveals that most gold particles are preferentially concentrated across from the inner nuclear membrane (ii, inner nuclear membrane outlined in white). Nucleus is denoted as N, vacuoles as V. Scale bars, 0.3 µm.

View larger version (67K): [in a new window]   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 PCUP1-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 PCUP1-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).

View larger version (87K): [in a new window]   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 PCUP1-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 PGAL1-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.

View larger version (25K): [in a new window]   Fig. 5. PMN-mediated degradation of Nvj1p is unperturbed in osh1 cells. (A,B) Starvation-induced degradation of Nvj1p-Myc is dependent on VAC8 and PEP4 but not ATG7 or OSH1. The turnover of a limited pool of Nvj1p-Myc during nitrogen starvation was analysed by immunoblot in isogenic wild-type, atg7 and vac8 cells (A) or osh1 and pep4 cells (B). The amount of Nvj1p-Myc remaining after 10 hours in SD-N was digitally quantified and compared with the level before starvation. Bar graphs represent the average percentage loss of Nvj1p-Myc from several independent experiments.

View larger version (45K): [in a new window]   Fig. 6. Cells depleted of Osh protein accumulate PMN structures under non-starving conditions. (A) Localization of Nvj1p-EYFP as a function of Osh protein depletion. An Osh depletion strain (osh1-osh7 PMET3-OSH2) and its parental wild-type background harboring PCUP1-NVJ1-EYFP were grown to low log phase in the presence or absence of methionine. To deplete all Osh protein, the Osh depletion strain was shifted into methionine-containing medium for 8 hours. Nvj1p-EYFP was induced for 1.5 hours with CuSO4, and its localization was monitored with respect to vacuolar membranes (red) and nuclear chromatin (blue). PMN structures increase in frequency upon the depletion of Osh protein (v,vi, arrows). (B) Kinetic analysis of Nvj1p-EYFP degradation in cells depleted of Osh protein. Starvation-induced degradation of a limited pool of Nvj1p-EYFP (arrows) was analysed by immunoblot in wild-type, pep4 and osh cells (depleted of Osh protein). Bar graph represents the average percentage loss of Nvj1p-EYFP from two independent experiments. Extract of cells lacking Nvj1p-EYFP is denoted C; a non-specific band cross-reacts above Nvj1p-EYFP on the blots. (C) Cells depleted of Osh protein (osh) show normal sorting and processing of aminopeptidase I precursor (prApe1p) into its mature form (mApe1p) by autophagy. Extracts of pep4 cells, which accumulate unprocessed prApe1p in the vacuole, were used as controls.