title: In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes.
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.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724.
authors.forename: Aman Y
authors.initials: AY
authors.lastname: Husbands
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
authors.forename: Vasudha
authors.initials: V
authors.lastname: Aggarwal
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 Howard Hughes Medical Institute, Baltimore, Maryland 21205.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Center for Plant Molecular Biology, University of Tübingen, 72076 Tübingen, Germany marja.timmermans@zmbp.uni-tuebingen.de.
authors.forename: Marja C P
authors.initials: MC
authors.lastname: Timmermans
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C493946:HD-Zip protein, Arabidopsis; D018398:Homeodomain Proteins; D014157:Transcription Factors