FIGURE 3.

Lhs1 requires ATP to associate with Kar2 and trigger nucleotide exchange. A, Lhs1 and His₆-Smt3-Kar2 were incubated in the presence and absence of ATP and loaded onto cobalt-NTA matrix. After washing, bound protein was eluted by proteolytic cleavage of the His₆-Smt3 using Ulp1 protease and analyzed on SDS-PAGE. B, fold-stimulation of the basal rate of MABA-ADP dissociation from Kar2 NBD (2 μm) induced by Lhs1 (2 μm) preincubated at the indicated concentrations of ATP. The release rates were measured with a stopped flow instrument. C, ATP-dependent interaction between Lhs1–2, Lhs1–3, and Lhs1–4 and Kar2. Quantitation of pulldown assays (see A) given as percent of wild-type Lhs1 bound to the Kar2 NBD. Error bars indicate the standard error (n = 4).