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.