Figure 5. Hxk1p interacts with Histone Deacetylase (Sir2) to repress filament specific genes.

A) The Hxk1p complex was purified by tandem affinity purification (TAP-tag) using anti- FLAG and Ni-nitrilotriacetic acid (Ni-NTA) agarose. Protein fractions of purified samples from 1st round with Anti-FLAG Agarose and 2nd round with Ni-NTA Agarose were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Open arrowheads indicate the component of the Hxk1p complex identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry. CAI-pYPB (control, CAI-4 transformed with pYPB-ADH1-pt, empty vector) and CAI-pYPB-HX-6HF (TAP tagged HXK1expressed under ADH1 p in CAI-4) are Spider medium induced cell extracts. B) Hxk1 interaction with Sir2 confirmed by Co-Immunoprecipitation. BW-pYPB-HX-6HF-SIR-HA and BW-pYPB strain cells were induced in Spider and crude extract isolated as described in Experimental procedures. Immunoprecipitation was carried out in two reactions with anti-HA-Agarose. The crude extract was used as an input. Immunoblotting was performed with anti-FLAG antibody. C) sir2 mutant showed hyperfilamentation similar to that of hxk1 mutant in inducing and non-inducing media. Upper panel shows the morphology of wild type, hxk1 mutant and sir2 mutant grown on YPD plates at 30°C for 3days and lower panel shows the morphology of wild type, hxk1 mutant and sir2 mutant grown on Spider plates at 37°C for 5 days.