FIGURE 2.

Residues conserved between Lhs1 and Sse1 are important for NEF activity. A, 10-fold serial dilutions of yeast strains CAY1171 (lhs1Δ sil1Δ) and CAY1172 (lhs1Δ sil1Δ ire1Δ) transformed to His⁺ using a vector control (vc), pCA715 (LHS1), or pCA715 carrying lhs1–2, lhs1–3, or lhs1–4 alleles. Cells were spotted onto control synthetic complete medium (SC) and medium containing FOA to counterselect against pCA716 (URA3 LHS1) that is present in the strains. Plates were incubated for 48 h at 30 °C. B, fold-stimulation of the basal rate of MABA-ADP dissociation from Kar2 NBD (2 μm) at the indicated concentrations of Lhs1 (♦) and derived mutants Lhs1–2 (■), Lhs1–3 (▲), and Lhs1–4 (●). Note that the data points for 4 μm Lhs1–2 and Lhs1–4 overlap. Proteins were preincubated with 3 mm ATP before measuring the release rates at 30 °C with a stopped flow instrument.