Prp8p binds ubiquitin in vitro. (A) Lysate from a yeast strain expressing triple HA-tagged Prp8p was prepared and incubated with ubiquitin-Sepharose or Sepharose beads in the presence of indicated amounts of free ubiquitin. The beads were washed, and the bound material was fractionated by SDS-PAGE and immunoblotted with αHA antibody. For comparison, unfractionated extract corresponding to 5% of the input material was analyzed in parallel. (B) Prp8p binds directly to ubiquitin in vitro. Recombinant Prp8p fragment corresponding to C-terminal residues 2143–2413 (including the Jab1/MPN domain) and C-terminal hexahistidine and HA tags was expressed and purified. The ability of this fragment to bind ubiquitin in vitro was analyzed as in A. (C) The Jab1/MPN domain of Prp8p is important for ubiquitin binding. Untagged recombinant Prp8p fragment that contained a wild-type or mutated Jab1/MPN domain (corresponding to the prp8-602 and prp8-603 alleles) was expressed in Escherichia coli. Ubiquitin binding was analyzed as in A, except that the binding was done at room temperature or 37°C and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999). (D) Mutations in Prp8p that do not affect ubiquitin binding. Untagged recombinant Prp8p fragment that contained a wild-type or mutated Jab1/MPN domain (corresponding to the prp8-1, prp8-28, and prp8-D144 alleles) was expressed in E. coli. Ubiquitin binding was analyzed as in A, except that the binding was done at room temperature or 37°C and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999). (E) An I44A mutation in ubiquitin diminishes ubiquitin binding by Prp8p fragment. Untagged recombinant Prp8p fragment that contained a wild-type Jab1/MPN domain was expressed in E. coli. Ubiquitin binding was analyzed as in A, except that binding was done with hexahistidine-tagged wild-type or I44A ubiquitin that was immobilized on Ni-NTA magnetic agarose resin and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999).
