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. 2012 Mar 19;196(6):713–725. doi: 10.1083/jcb.201110090

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© 2012 Kim et al.

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Figure 4. — Mutational analysis of the roles of individual regulatory bonds in autonomous reoxidation of Ero1p. (A) Ero1pc-C150A-C295A, Ero1pc-C90A-C349A, or Ero1pc-C143-C166A was reduced (red) by incubation with reduced Trx1 followed by Trx1 depletion and the oxidation (ox) state analysis. (B) The fraction of reduced Ero1p was calculated by the ratio of the band intensity of reduced Ero1p species to the sum of fully reduced and fully oxidized band intensities. (C) Mutational analysis of the roles of individual regulatory bonds in reduction by reduced Pdi1p of Ero1pc-C100A-C105A. The respective mutants were incubated with a 20-fold excess of reduced Pdi1p followed by the oxidation state analysis (the result of Ero1pc-C100A-C105A was reproduced from Fig. 3 B). (D) The fraction of reduced Ero1p determined as in B reveals that the greatest rate enhancement occurs as a consequence of elimination by mutation of the C150-C295 disulfide bond.