Deregulation of HMG proteins induces necrosis. (A) H3WT cells were transformed with control vector or a vector expressing one of the five indicated yeast HMG genes from the GAL1 promoter. Equal numbers of cells were 5-fold serially diluted and spotted to SC-uracil plates containing glucose (repressive) or SC-uracil galactose (activating) media and incubated at 30°C for 4 days. (B-D) Cells containing control vector and either the galactose-inducible Hmo1 (B), Nhp10 (C) or Ixr1 (D) expression vectors were grown to log phase in raffinose media before serially diluting 5-fold and spotting to SC-uracil glucose plates. The remaining cultures were treated with 2% galactose for the indicated times before washing and spotting as described. Plates were incubated for 3 days at 30°C. (E) H3WT cells carrying control, GAL1-HMO1, GAL1-NHP10, or GAL1-IXR1 expression vectors were grown to log phase in SC-uracil raffinose media, induced with 2% galactose for 24 hours, stained with Annexin V and PI, and then analyzed by confocal microscopy. Scale bar indicates 10 μm. (F) The strains from (B-D) were induced with 2% galactose for 1 hour before preparing whole-cell extracts. 30 μg of extract was resolved by SDS-PAGE and immunoblotted with α-HA antibody to detect the HA-tagged Hmo1, Nhp10 and Ixr1. (G) Model for TORC1 signaling and regulation of HMG chromatin association. Details in the text. Gal, galactose; PI, propidium iodide; ura, uracil, vect, vector.