title: Purification of low-abundance Arabidopsis plasma-membrane protein complexes and identification of candidate components.
abstract: 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.
authors.affiliation: Department of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, St Paul, MN 55108, USA.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Qi
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Katagiri
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; C089426:RPS2 protein, Arabidopsis