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. 2016 Jul 15;27(14):2213–2233. doi: 10.1091/mbc.E16-03-0177

FIGURE 8:

FIGURE 8:

Cells lacking mih1∆ provide a sensitized background for assessment of Hsl1 function. (A) Simultaneous absence of Mih1 and Hsl1 is lethal. Strains of the BY4741 lineage expressing either Cdc10-mCherry (GFY-42) or Cdc11-mCherry (GFY-58) as indicated and carrying the wild-type HLS1 and/or MIH1 loci or carrying an hsl1∆ mutation (GFY-1156 and GFY-1157), an mih1∆ mutation (GFY-1881 and GFY-1882), or both (GFY-1737 and GFY-1738), and harboring a wild-type copy of the HSL1 gene on a URA3-marked CEN plasmid (pGFY-316-IVL924) were incubated for 2 d at 30˚C on medium lacking uracil (left) or containing 5-FOA (right) to select for the loss of the covering plasmid. (B) Toxicity of overexpressed Hsl1 is ameliorated by eliminating its kinase activity, revealing that the 611–950 segment combined with the KA1 domain is necessary and sufficient to compete for the function of endogenous Hsl1. Wild-type cells (BY4741, leftmost pair) or an otherwise isogenic mih1∆ derivative (GFY-1652, rightmost pair) were transformed with plasmids overexpressing from the GAL1/10 promoter eGFP alone (pGF-IVL391), full-length Hsl1 (fused to eGFP; pGF-IVL302), or the other indicated Hsl1 derivatives (pGF-IVL694, pGF-IVL689, pGF-IVL932, pGF-IVL642, pGF-IVL643, pGF-IVL929, pGF-IVL893A, pGF-IVL893B, pGF-IVL863, pGF-IVL894B, pGF-IVL895, pGF-IVL930, pGF-IVL896, pGF-IVL931, pGF-IVL644, pGF-IVL645, and pGF-IVL646), grown overnight at 30°C in liquid SD-Leu medium containing 2% raffinose and 0.2% sucrose, spotted onto agar plates containing SD-Leu (left) or SGal-Leu (right), and incubated at 30°C for 3 d. In two constructs, C2Lact domain was fused to the C-terminus of the Hsl1(1–374)-GFP or Hsl1(1–374; D239A) fragment, as indicated. Red asterisks, overexpressed fragments containing both the 611–950 segment and the KA1 domain cause an Hsl1-deficient phenotype in mih1∆ cells. (C) Hsl1 kinase-dead (D239A) allele localizes normally. Cells of strain GFY-42 expressing both Cdc10-mCherry from its endogenous locus and a full-length, catalytically inactive Hsl1 allele, Hsl1(D239A) (tagged with eGFP), from a plasmid (pGF-IVL693) were visualized by fluorescence microscopy. Scale bar, 2 μm.