Husbands, A. Y., Aggarwal, V., Ha, T., Timmermans, M. C. (August 2016) In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes. Plant Cell, 28 (8). pp. 1783-94. ISSN 1532-298X (Electronic)1040-4651 (Linking)
Abstract
Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics organism description > plant bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression |
| CSHL Authors: | |
| Communities: | CSHL labs > Timmermans lab |
| Depositing User: | Matt Covey |
| Date: | August 2016 |
| Date Deposited: | 29 Nov 2016 17:09 |
| Last Modified: | 19 Jun 2017 14:54 |
| PMCID: | PMC5006705 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/33904 |
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