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.