Figure 5.

Ankyrin (Ank) repeats of Osh1p specify targeting to the NV junction. (A) Structure of Osh1p and of the constructs examined (plasmids pTL321-325). Osh1p can be divided into four regions: Ank, containing three ankyrin repeats; PH, PH^Osh1 (in some constructs replaced by PH^spectrin); HD, a domain predicted to contain helical regions; and OBD, oxysterol binding domain. The constructs are tagged with GFP (“G”). (B–F) Triple label fluorescent micrographs of live log phase Δosh1 cells (TLY221) expressing the indicated GFP-tagged constructs and stained with FM4-64 (red) and DAPI (blue) as in Figure 4. Arrows indicate typical NV junctions. The same localizations were seen in wild-type cells (our un published results). The NV junctions are relatively small, and so are not always visible in any given focal plane. When cells expressing GFP-Osh1p were counted the percentage of cells showing clear staining in a linear structure between opposed nuclei and vacuoles was typically 85–95%. The exceptions were almost always small daughter cells where the vacuole is still highly fragmented and presumably the NV junction has yet to properly form. A comparable frequency of NV junction structures was seen with all constructs containing the N-terminal ankyrin repeat domain of Osh1p (G) Δvac8 cells (TLY232) expressing pTL324 treated as in B–F except FM4-64 staining was for 45 min, followed by a 3-h chase to examine transfer of vacuolar material to daughter cells, confirming that loss of Vac8p leads to reduced vacuolar segregation into daughter cells (arrowhead), and highly fragmented vacuoles in some cells (double arrowhead) (Fleckenstein et al., 1998; Pan and Goldfarb, 1998; Wang et al., 1998).