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

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• 作者：Maarten Dedecker ; Jelle Van Leene ; Nancy De Winne…
• 刊名：Plant Molecular Biology
• 出版年：2016
• 出版时间：June 2016
• 年：2016
• 卷：91
• 期：3
• 页码：341-354
• 全文大小：841 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Plant Sciences
  Biochemistry
  Plant Pathology
  
• 出版者：Springer Netherlands
• ISSN：1573-5028
• 卷排序：91

文摘

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 re-occurrence 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 SUBUNIT 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-of-the-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.KeywordsAnaphase promoting complexCYCLIN-DEPENDENT KINASE DOryza sativa (rice)Protein–protein interactionsTandem affinity purification coupled to mass spectrometry (TAP–MS)