FIGURE 9:

Preventing APC-mediated degradation and blocking nuclear import of the 611–950 fragment of Hsl1 markedly enhance its ability to confer an Hsl1-deficient phenotype by displacing endogenous Hsl1 from the bud neck. (A) Wild-type cells (BY4741, leftmost pair) or an otherwise isogenic mih1∆ derivative (GFY-1652, rightmost pair) were transformed with either empty vector (pRS315) or derived plasmids overexpressing from the GAL1/10 promoter either GST alone (pGF-IVL914) or the other indicated fragments of Hsl1 fused to GST (pGF-IVL911, pGF-IVL916, pGF-IVL912, pGF-IVL917, pGF-IVL913, and pGF-IVL918), which were also tagged with a FLAG epitope at the end opposite from that fused to GST and then propagated and tested for growth as described in Figure 8B. (B) Strain GFY-1739, expressing both Cdc10-mCherry and Hsl1-GFP from their endogenous loci, was transformed with the same plasmids as in A, grown overnight to saturation in SD-Leu medium containing 2% raffinose and 0.2% sucrose, backdiluted in SD-Leu medium containing 2% galactose, incubated for 4–5 h at 30°C, and then examined by fluorescence microscopy. Arrowheads, mCherry-marked septins deposited at ectopic locations. (C) Representative cells in cultures of the same strains as in B, numbered 1–8 for clarity, that were grown overnight in SGal-Leu medium to saturation and then examined by DIC. Images were scaled identically; scale bar, 2 μm.