An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation

Abstract

SummaryTandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis. In our newly generated TAPa tag we have replaced the tobacco etch virus (TEV) protease cleavage site with the more specific and lowtemperature active rhinovirus 3C protease site. In addition, the second purification step can now be performed through two different affinity tags: a six His repeat or nine copies of a myc repeat. To examine our purification procedure we generated a C-terminal fusion between the TAPa tag and CSN3, a component of the multiprotein COP9 signalosome (CSN) complex. Subsequent analysis showed that CSN3-TAPa could rescue a csn3 mutant, and that the components of the CSN complex could be co-purified with CSN3-TAPa. As part of our long running interest in light signaling in Arabidopsis we have generated Arabidopsis transgenic lines harboring, both N-terminal and C-terminal TAPa fusions of many different light signaling pathway regulators. Molecular characterization of these transgenic lines showed fusion expression in 88% of the genes analyzed and that this expression is largely independent of the fusion orientation. Mutant complementation analysis showed that most of the TAPa fusions analyzed retained function of the wild-type proteins. Taken together, the data demonstrate the suitability of the TAPa system to allow efficient multi-protein complex isolation from stably transformed Arabidopsis.

Auxin Responses in Mutants of the Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC9 Signalosome

“…To obtain a homogeneous set of csn mutants for such a transcriptprofiling experiment, we examined mutants for CSN3, CSN4, and CSN5 in the Arabidopsis ecotype Columbia (Col-0) from the SALK T-DNA insertion mutant collection ( Fig. 1A; Gusmaroli et al, 2004;Dohmann et al, 2005;Rubio et al, 2005). Like all previously identified CSN subunit loss-of-function alleles, these csn3, csn4, and csn5ab (csn5a-2 csn5b-1) mutants have a constitutive photomorphogenic phenotype, arrest growth at the seedling stage, and are deficient in the deneddylation of Arabidopsis cullins as shown by western blots with antibodies directed Figure 1.…”

“…The following T-DNA insertion mutant alleles were identified and obtained from the Nottingham Arabidopsis Stock Centre: csn3-1 (Salk_000593), csn3-2 (Salk_106465), csn4-1 (Salk_043720), and csn4-2 (Salk_053839; Rubio et al, 2005). The csn3 alleles were genotyped using the oligonucleotides CSN3-ATG-FW and CSN3-RV to detect the wild-type gene, and LBb1 and CSN3-E9-RV to test for the presence of the T-DNA insertion.…”

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

“…Moreover, complementation was achieved at the protein level, with chloroplast proteins accumulating at wild-type levels ( Figure 1B). We also employed other C-terminal tagging systems, including a polyhistidine tag (His 6 ) and a tandem affinity tag (Rubio et al, 2005); whereas partial or complete phenotypic complementation was achieved, the isolated Clp complex was never sufficiently pure, as judged by MS/MS analysis. Therefore, these efforts will not be further discussed.…”

Mutation of OsNaPRT1 in the NAD Salvage Pathway Leads to Withered Leaf Tips in Rice

“…Total protein extraction from leaf tissue was performed as previously described (Rubio et al, 2005). Two to three leaves were ground into powder in liquid nitrogen and transferred into a 1.5-mL centrifuge tube.…”