Figure 6

Tetrameric formation of Sre1-CTD. (A) The N-terminus of the α1 helix contains two aromatic residues Trp702/Tyr703 that are highly conserved in related species and are shown as sticks. (B) AUC-SV analysis of the Sre1-CTD oligomeric mutants. The AUC-SV analysis of the WT, PPP+4 mutant and WY mutant of Sre1-CTD supported that the conserved WY motif contributed to the tetrameric formation. (C) SLS analysis of the Sre1-CTD oligomeric mutants. SLS characterization of WY mutant produced two peaks corresponding to 57.5 kDa and 30 kDa, respectively, suggesting less stable dimer formation. (D) WT, sre1Δ and sre1 WY mutant cells (5 000, 1 000, 200, 40 and 8 cells) were grown on rich medium (3 days) or rich medium containing cobalt chloride (1.6 mM; 6 days). (E) Western blot of whole-cell lysates from WT, sre1Δ and sre1 WY cells grown in the presence or absence of oxygen for 3 h with anti-Sre1 serum. P and N denote Sre1 precursor and nuclear forms, respectively. Asterisks indicate cross-reacting proteins. (F) Western blot of membrane fractions from WT and WY mutant cells with or without hrd1⁺ probed with anti-Sre1 serum or anti-Dsc5 serum.