The oligomeric state of She2. (A) Cross-linking analysis of She2. SHE2-1/2TAP was expressed in wild-type cells, and purified She2 was treated with 40 mM EDC for 1 h at room temperature, separated by 4–15% SDS-PAGE, and probed with anti-She2 antibody. Note that She2 appears as a doublet in the absence of EDC due to oligomerization of wild-type and TAP-tagged She2. (B) She2 oligomerization is not concentration dependent. SHE2-1/2TAP was expressed from a high-copy plasmid in she2Δ cells, and cross-linking analysis was performed with purified She2 diluted to various concentrations. (C) She2 oligomerization does not require RNA binding. SHE2-1/2TAP was expressed in she2Δ, cells and U3 RNA expression was induced. Half of the cell extract was treated with 0.3 mg/ml RNaseA, and purified She2 from each cell extract was cross-linked. (D) Oligomerization is inhibited by mutations in the upper uncharged surface of She2. 1/2TAP-tagged SHE2 mutants (N36S, R63K, T47Y, and L130Y) were expressed in she2Δ cells, and the purified proteins were cross-linked and analyzed as described in A. (E) Analytical ultracentrifugation of She2. Purified wild-type She2 was diluted to 0.15 mg/ml (5.3 µM), 0.53 mg/ml (18.7 µM), 1.13 mg/ml (39.9 µM), and 1.90 mg/ml (67.1 µM), then subjected to analytical ultracentrifugation. A direct boundary modeling program from individual datasets using model-based numerical solutions to the Lamm equation was used to obtain data shown for the normalized continuous sedimentation coefficient, c(s), distribution plot. At 0.15 mg/ml, the weighted mean value of S obtained through integration of the c(s) curve was 5.95S.