Figure 5
From: C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex
Characterization of Spt7 Form3. A. YCp88-myc was transformed into BY4741 (lane 1, ctrl) and YCp88-myc-SPT7 into BY4741 (lane 2). Crude protein extracts were prepared and Western blotted with anti-myc antibody. The mobility of a 160 kDa molecular weight marker is indicated. Spt7Form3 is marked as F3. B. BY4741 (wild-type, right panel) and BY3218 (spt7Δ0, left panel) were transformed with YCpDed-TAP-Flag (lanes 1 and 3) or YCpDed-TAP-Flag-SPT7 into (lanes 2 and 4). Extracts were prepared by grinding 1 litre of cells in liquid nitrogen, and subjected to TAP purification. 20 μl of the peak fractions were blotted with anti-Flag antibody. C. Gel filtration chromatography of Spt7 from crude yeast extracts. 8.5 mg of protein extract was prepared from yeast strain BY4741 containing YCpDed-TAP-Flag-SPT7 and applied to a Superose 6 HR10/30 column (Pharmacia). 500 μl fractions were collected, precipitated with 10% trichloroacetic acid, separated on a 5% SDS-polyacrylamide gel and Western blotted with anti-Flag antibody. Relative amounts of each of the three forms as determined by densitometry are shown as are the peak fraction for elution of the indicated molecular mass standards. D. Wild-type (4741; lane 9) or strains containing C-terminally TAP-tagged Gcn5, Ada2, Ubp8, Taf5, Spt3 and Spt8, expressing Flag-Spt7 (lanes 3–8) were grown in YP media containing 2% glucose to an OD600 = 2.0. Twenty-five milligrams of crude lysate was purified on IgG agarose. Bound protein was separated by 5% SDS-PAGE and Flag-Spt7 forms detected with anti-Flag antibody. To demonstrate the position of the three Spt7 forms lanes 1 and 2 are extracts of BY4741 transformed with YCpDed-Flag-SPT7 that have been purified on Sepharose 4B (-'ve) or anti-Flag antibody coupled to Sepharose (Spt7) then Western blotted with anti-Flag antibody. The positions of molecular mass markers, Spt7SAGA (SA), SPT7SLIK (SL) and Spt7Form3 (F3) are indicated.