Purification of low‐abundance Arabidopsis plasma‐membrane protein complexes and identification of candidate components

Authors:
Publisher website
Google Scholar
Abstract
Summary: Purification of low‐abundance plasma‐membrane (PM) protein complexes is a challenging task. We devised a tandem affinity purification tag termed the HPB tag, which contains the biotin carboxyl carrier protein domain (BCCD) of Arabidopsis 3‐methylcrotonal CoA carboxylase. The BCCD is biotinylated in vivo, and the tagged protein can be captured by streptavidin beads. All five C‐terminally tagged Arabidopsis proteins tested, including four PM proteins, were functional and biotinylated with high efficiency in Arabidopsis. Transgenic Arabidopsis plants expressing an HPB‐tagged protein, RPS2::HPB, were used to develop a method to purify protein complexes containing the HPB‐tagged protein. RPS2 is a membrane‐associated disease resistance protein of low abundance. The purification method involves microsomal fractionation, chemical cross‐linking, solubilization, and one‐step affinity purification using magnetic streptavidin beads, followed by protein identification using LC‐MS/MS. We identified RIN4, a known RPS2 interactor, as well as other potential components of the RPS2 complex(es). Thus, the HPB tag method is suitable for the purification of low‐abundance PM protein complexes.

This publication has 81 references indexed in Scilit: