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First published online 15 August 2006
doi: 10.1242/jcs.03093

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Structure and function of nucleus-vacuole junctions: outer-nuclear-membrane targeting of Nvj1p and a role in tryptophan uptake

Department of Biology, University of Rochester, Rochester, NY 14627, USA

View larger version (57K): [in a new window]   Fig. 1. Nvj1p can be divided into four domains based on sequence homology across the Saccharomyces family. (A) Alignment of S. cerevisiae Nvj1p and its orthologs in closely related and divergent Saccharomyces species. Amino acid sequences were obtained from the Saccharomyces Genome Database (www.yeastgenome.org). The S. kluyveri ortholog of Nvj1p was identified through a tblastn search of contig 02.1344 (accession AACE02000001). Sequences were aligned using the ClustalX program (Thompson et al., 1997). Boxes were drawn around regions of sequence similarity in the Saccharomyces Nvj1p family. (B) Graphical view of residues in Nvj1p bearing similarity or identity across the Saccharomyces Nvj1p family. ClustalX conservation scores were calculated for each column of the alignment in 1A, and residues bearing similarity (50-74% homology, open circles) or near identity (75-100%, shaded diamonds) were aligned onto a linear map of Nvj1p. These residues sorted independently into four regions, as indicated. (C) Kyte-Doolittle hydropathy plot of the four conserved regions of Nvj1p. Region I at the N-terminus shows an area of considerable hydrophobicity, while region II coincides with the membrane-spanning domain of Nvj1p.

View larger version (41K): [in a new window]   Fig. 2. Tripartite signals mediate the targeting of Nvj1p to NV junctions. (A) Region IV comprises the Vac8p-binding domain of Nvj1p. Full-length (FL) Nvj1p-EGFP and a C-terminal truncation (293-321aa) lacking the most conserved residues of region IV were localized in vac8- and VAC8+ (nvj1-) cells. Nuclear chromatin (blue) and vacuole membranes (red) were co-stained with Hoechst and FM4-64, respectively. Interactions between Nvj1p-EGFP and Vac8p on FM4-64-stained vacuoles create NV junctions, which appear yellow in the overlay. The C-terminal truncation of Nvj1p does not form NV junctions and instead localizes uniformly across the nuclear envelope. (B) Region I plays a role in anchoring Nvj1p to the perinuclear ER. N-terminal truncations preceding (1-6aa) or succeeding (1-26aa) the hydrophobic sequence of region I, or eliminating the N-terminus of Nvj1p entirely (1-86aa), were localized in vac8- and VAC8+ (nvj1-) cells. Nuclear chromatin (blue) and vacuole membranes (red) were co-stained as above. EGFP-tagged truncations lacking region I (denoted by a horizontal bar) escape into the cortical ER and form aberrant vacuole-associated membrane junctions in the cytosol (see arrows) that appear detached from the nucleus. (C) Region I and the membrane-spanning region of Nvj1p (region II) cooperatively sort to the perinuclear ER. Nvj1p fragments tagged on their C-terminus with EGFP were localized similarly to above. The membrane-spanning region of Nvj1p (residues 87-120, denoted by a shaded box) targets ER membranes but localizes exclusively to the perinuclear ER in conjunction with region I (denoted by a horizontal bar). Replacing the transmembrane region of Nvj1p (between residues 90-121) with the first transmembrane segment of Ste2p causes Nvj1p to spread into the cortical ER and form aberrant junctions with vacuoles in the cytosol (see arrows). Numbers in diagram correspond to amino acid position.

View larger version (90K): [in a new window]   Fig. 3. Blocking the N-terminus of Nvj1p promotes extranuclear junction formation by separation and extension of the ONM. (A) GFP-Nvj1p localizes throughout the perinuclear and cortical ER in vac8- cells, and accumulates in extranuclear junctions between ER-like membranes and cytoplasmic vacuoles when bound to Vac8p. Vacuole membranes (red) were stained with FM4-64 and overlayed. Images were collected by Xiaozhou Ryan. (B) Immunogold localization of GFP-tagged Nvj1p by electron microscopy. GFP-tagged Nvj1p reporters were induced prior to cryofixation and immuno-EM analysis as described in Materials and Methods. Nvj1p-GFP, a functional reporter, localizes to NV junctions. Colloidal-gold-conjugated antibodies against GFP (asterisk) label the surface of nuclei at NV junctions. By contrast, GFP-Nvj1p (a non-functional reporter) forms extranuclear junctions with vacuoles. Here, colloidal-gold-conjugated antibodies against GFP (asterisk) label an extension of the ONM (black arrows) that is sandwiched between two vacuole lobes. The ONM is separated from the INM (denoted by white arrowheads), forming an extensive void in the perinuclear lumen. N, nuclei; V, vacuoles. Bars, 0.3 µm.

View larger version (25K): [in a new window]   Fig. 4. Region II of Nvj1p sequesters Tsc13p. (A) The membrane-spanning domain of Nvj1p (region II) is responsible for recruiting Tsc13p-EYFP. Full-length (FL) or N- and C-terminal truncations of Nvj1p were overexpressed in nvj1- cells and tested for the ability to sequester Tsc13p-EYFP from cortical ER compartments to the perinuclear ER. Nuclear chromatin (blue) and vacuole membranes (red) were co-stained with Hoechst and FM4-64, respectively. Regions flanking either side of the membrane-spanning domain of Nvj1p (denoted by a shaded box) proved dispensable for the sequestration of Tsc13p-EYFP to the nuclear envelope. Numbers correspond to amino acid positions. (B) The transmembrane region of Nvj1p (aa 85-120) is sufficient to co-immunoprecipitate Tsc13p-EYFP in vivo. Detergent-extracted lysates of cells expressing Tsc13p-EYFP and 3HA-tagged Nvj1p(85-120aa) or empty vector were prepared as described in Materials and Methods. Nvj1p(85-120aa)-3HA was immunoprecipitated (IP) with anti-HA conjugated agarose beads.

View larger version (24K): [in a new window]   Fig. 5. Region III of Nvj1p is necessary and sufficient to bind Osh1p. (A) Deletion of the most conserved residues in region III abrogates the Nvj1p-dependent sequestration of GFP-Osh1p to the nuclear envelope. Full-length (FL) or N- and C-terminal truncations of Nvj1p were overexpressed in nvj1- cells and tested for the ability to sequester GFP-Osh1p from soluble and Golgi-associated pools to the nuclear surface. Conserved residues in region III of Nvj1p (aa 130-176) proved necessary for GFP-Osh1p sequestration. Numbers correspond to amino acid positions. (B) Residues 130-177 of Nvj1p are sufficient to co-immunoprecipitate GFP-Osh1p in vivo. Lysates of cells expressing GFP-Osh1p and 3HA-tagged Nvj1p(130-177aa) or empty vector were prepared as described in Materials and Methods. Nvj1p(130-177aa)-3HA was immunoprecipitated (IP) with anti-HA conjugated agarose beads.

View larger version (29K): [in a new window]   Fig. 6. Overexpression of Nvj1p, but not a mutant form lacking Osh1p-binding activity, confers a tryptophan transport-related growth defect. (A) Cells overexpressing Nvj1p, but not a mutant form lacking the Osh1p-binding domain, grow poorly on media containing limiting concentrations of tryptophan at low temperature. Cells harboring PCUP1-NVJ1, PCUP1-NVJ1(130-176aa)-myc, or empty vector were induced and replica-plated onto media containing limiting (15 µg/ml) or excess (40 µg/ml) tryptophan as described in Materials and Methods. (B) The cold-sensitive growth phenotype of Nvj1p-overexpressing cells is suppressed by low- and high-affinity tryptophan permeases. Cells harboring PCUP1-NVJ1 and either pTAT1, pTAT2, or empty vector were induced and replica-plated onto media containing limiting (15 µg/ml) or excess (40 µg/ml) tryptophan as above. (C) NV junctions are not necessary to confer tryptophan transport defects in Nvj1p-overexpressing cells. Growth curves of vac8- cells expressing PCUP1-NVJ1 (open squares), PCUP1-NVJ1(130-176aa)-myc (black triangles), or empty vector (gray circles) were determined in liquid media containing limiting (15 µg/ml) or excess (40 µg/ml) tryptophan at 25°C using a Bioscreen C Analyzer (see Materials and Methods).

View larger version (17K): [in a new window]   Fig. 7. Native levels of Nvj1p are mildly deleterious for growth in tryptophan-depleted media. nvj1- and its isogenic parental (NVJ1+) tryptophan auxotrophic strain (trp1) were grown in rich media to log-phase and shifted into SC media containing limiting (15 µg/ml) or depleted (5 µg/ml) concentrations of tryptophan. Cell growth was monitored at 25°C using a Bioscreen C Analyzer (see Materials and Methods). Error bars were calculated from two independent trials performed in duplicate.

View larger version (18K): [in a new window]   Fig. 8. Molecular model of NV junctions. Nvj1p is restricted to the outer nuclear membrane, which is continuous with the perinuclear ER. The membrane-spanning domain (II) of Nvj1p interacts with Tsc13p in the ER membrane. An adjacent cytosolic region (III) associates with Osh1p. The C-terminal tail of Nvj1p (IV) binds to Vac8p, which is acylated to the vacuole membrane. The hydrophobic N-terminus of Nvj1p (I) may link the inner- and outer-nuclear membranes either by binding an unknown factor (X) in the INM (panel i), or by inserting directly into the INM by spanning across the perinuclear lumen (panel ii).

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