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. 2016 Aug 4;35(19):2139–2151. doi: 10.15252/embj.201694082

Figure EV6. Purification of Hrr25 and Hrr25:Mam1 for ATPase assays and modeling of Mam1 Arg131 interaction with nucleotide and CK1‐7.

Figure EV6

  1. SDS–PAGE analysis of purified Saccharomyces cerevisiae Hrr251–394 and Hrr251–394:Mam187–191 complexes used for ATPase assays.
  2. Ni2+ affinity pull‐down of co‐expressed His6‐Hrr251–394 K38R:Mam187–191 complexes with mutations to Mam1 in interfaces #1 or #2. Except for R131A, all mutations result in a loss of co‐purification of Mam1, indicating a loss of binding, and a significant reduction in the level of soluble Hrr251–394 K38R.
  3. Close‐up views of the Hrr25 active site in the structure of S. cerevisiae Hrr251–394:Mam187–191. Mam1 R131 extends toward the ATP‐binding pocket, and while it would not clash with a bound nucleotide (center panel), it would clash with the aminoethyl group of bound CK1‐7 (right panels).