This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Nonessential portions of the yeast nucleus are targeted to the vacuole and degraded by piecemeal microautophy of the nucleus (PMN) (Roberts et al., 2003; Goldfarb, 2003). During PMN, teardrop-like nuclear blebs are engulfed by invaginations of the vacuole membrane, pinched into the vacuole lumen, and degraded by acid hydrolases. PMN occurs in the context of nucleus-vacuole (NV) junctions, which are Velcro-like patches formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p on the outer nuclear membrane (Kvam and Goldfarb, 2004, Kvam et al., 2005). We are applying both standard electron tomography of high pressure frozen, freeze-substituted material and as cryo-electron tomography of frozen hydrated cryosections to study the NV junction in wild-type and mutant strains, including cells expressing the N-terminal truncation of Nvj1p and strains with an expanded N-terminal domain. These studies should provide a unique and informative approach for a detailed structural analysis of this junction and should afford the resolution necessary to identify and characterize putative cross-bridges between membranes. Our initial tomographic studies used yeast that had been high pressure frozen, freeze-substituted and embedded in plastic. These preparations showed that the separation between the inner and outer nuclear envelope is less at the NV junctions than elsewhere, and that the spacing expands in regions away from the NV junction, supporting the hypothesis that Nvj1p may span the perinuclear lumen and connect the inner and outer nuclear membranes. We have also begun to image samples in the frozen-hydrated state by cutting cryosections of yeast that have been high pressure frozen. Cryo-electron tomography of the sections is being preformed to analyze the NV junction in a state that is close to its native condition. Preliminary work shows promising results from profiles of NV junctions in cryosections of wild-type yeast. This emerging technology has the advantage of imaging the NV junctions that are fully hydrated, unfixed and unstained. Combining electron tomography with a genetic approach should yield a better understanding of the NV junction in yeast in particular, and contribute to our understanding of the proteins that connect membrane systems in general.
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