Figure 2. Functional importance of the NTR of yeast Isw1 in vivo.
(A) Successive N-terminal truncation mutants of Isw1. Note that Isw1ΔNTR lacked the entire N-terminus up to the first seven residues of AcidicN (Figure 1E). (B) Complementation assay with Isw1ΔppHSA. A yeast strain lacking ISW1, ISW2 and CHD1 (TKO) was transformed with Isw1 derivatives under control of promoters of varying strengths. In comparison to a strain lacking only ISW2 and CHD1 (DKO), Isw1WT fully complemented the growth phenotype at elevated temperatures (37°C). In contrast, Isw1ΔppHSA did not complement at any expression level. Results for other Isw1 variants can be found in Figure 2—figure supplement 1. Growth was assessed by spotting tenfold serial dilutions of liquid cultures.
Figure 2—figure supplement 1. Complementation assay with N-terminal truncation variants of Isw1.
(A–D) Growth assays as in Figure 2B. Expression levels were estimated by Western analysis (see panel E). Results in D (30°C and 37°C) are replotted from Figure 2. (E) Exemplary Western blot to determine relative expression levels (tabulated in A–D) using an Anti-TAP antibody. TAP-tagged Isw1 variants under control of the indicated promoter were expressed. Their expression level was normalized against genomically TAP-tagged wild-type Isw1. Errors are minimal and maximal values of two technical replicates. H3 served as a loading control.