Figure 4.
ACHT1 Reacts with 2-Cys Prx in Plants.
(A) Intermolecular disulfide complexes of ACHT1 and its redox protein partners. Immunoblot assay showing the intermolecular disulfide complexes trapped in plants expressing the catalytic active ACHT1 (DT) or the monothiol mutant ACHT1C35S (MT). Proteins were electrophoresed under nonreducing (NR) or reducing (R) conditions, and complexes containing ACHT1 were identified by immunoblot assays using anti-HA-specific antibodies. The major intermolecular disulfide complexes of ACHT1, which were identified by MS, are marked with an asterisk.
(B) Reciprocal immunoprecipitation verified the ACHT1–2-Cys Prx interaction. Immunoblot assay of proteins immunoprecipitated with anti-HA (ACHT1 IP) or anti-2-Cys Prx (Prx IP) affinity matrixes or with nonspecific matrix (Control IP) from plants expressing ACHT1C35S. Purified proteins were run under reducing conditions and blotted with antibodies specific to the HA-tag of ACHT1 (αHA) or to 2-Cys Prx (αPrx).