Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

Abstract

Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on reoccurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUB-UNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-ofthe-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.

Recent Trends in Plant Protein Complex Analysis in a Developmental Context

“…This example demonstrates the benefits of performing AP/MS experiments on specific organs or even sub-organ tissues as well as the need to transfer AP/MS to other model systems like certain crop species, which might be better suited, as was illustrated by the maize leaf. Similar complex purification strategies have been demonstrated for the rice leaf ( Dedecker et al, 2016 ). The use of alternative model systems might also be required when specialized developmental processes are explored.…”

Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures

“…For immunoblot analysis, samples were centrifuged as described in Dedecker et al . (). Protein concentrations were determined by Bradford assay (Bio‐rad, Hercules, CA, USA).…”

“…Crude protein extracts were prepared with 15 mL of extraction buffer [25 mM Tris-HCl pH 7.6, 15 mM MgCl 2 , 150 mM NaCl, 15 mM p-nitrophenyl phosphate, 60 mM b-glycerophosphate, 0.1% NP-40, 0.1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 1 lM E64, EDTA-free Ultra Complete tablet Easypack (1/10 mL; Roche Diagnostics, Brussels, Belgium), 5% ethylene glycol] with an Ultra-Turrax T25 mixer (IKA Works, Wilmington, NC, USA) at 4°C. For immunoblot analysis, samples were centrifuged as described in Dedecker et al (2016). Protein concentrations were determined by Bradford assay (Bio-rad, Hercules, CA, USA).…”

“…Total protein extract (30 lg) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 0.75 mm 12% Mini-PROTEAN â TGX TM precast gels; Bio-Rad), and immunoblot analysis was executed as described in Dedecker et al (2016). Bound antibody was detected using detection substrate (Western Lightning Plus-ECL; Perkin Elmer, Waltham, MA, USA) and X-ray films (Amersham hyperfilm TM ECL; GE Healthcare, Wauwatosa, WI, USA).…”

“…Proteolysis and peptide isolation were basically executed as described in Dedecker et al (2016). NuPAGE gels with the protein samples were de-stained twice in HPLC-grade water (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h and transferred to reducing buffer (6.66 mM DTT, 50 mM NH 4 HCO 3 in HPLC-grade water) for 40 min.…”