title: Identification of Interacting Motifs Between Armadillo Repeat Containing 1 (ARC1) and Exocyst 70 A1 (Exo70A1) Proteins in Brassica oleracea.
abstract: In order to identify the functional domains which regulate the interaction between the self-incompatibility proteins armadillo repeat containing 1 (ARC1) and exocyst 70 A1 (Exo70A1) in Brassica oleracea, fragments containing selected motifs of ARC1 (ARC1210, ARC1246, ARC1279, ARC1354) and site-specific mutants with substitutions at possible interaction sites (ARC1354m, ARC1664m) were PCR amplified and inserted into pGADT7, while coding sequences from Exo70A1 (Exo70A185, Exo70A1) were subcloned into pGBKT7. The interactions between the protein products produced by these constructs were then analyzed utilizing a yeast two-hybrid system. Our data indicate that both ARC1210 and ARC1246 interact strongly with Exo70A185 and Exo70A1, while ARC1279, ARC1354, ARC1354m and ARC1664m exhibited a weak interaction, indicating that the recognition sites are located within the 210 N-terminal amino acids of ARC1 and the 85 N-terminal amino acids of Exo70A1. This was further verified by GST pull-down analysis. This supports a model in which the N-terminal leucine zipper of ARC1 and the first 85 N-terminal amino acids of Exo70A1 mediate the interaction between these two proteins. Bioinformatic and phylogenetic analysis demonstrated that these motifs were highly conserved across different species, indicating that the interaction characterized in B. oleracea may operate in a wide array of cultivars.
authors.affiliation: College of Agronomy and Biotechnology, Southwest University, Chongqing, 400716, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: College of Agronomy and Biotechnology, Southwest University, Chongqing, 400716, China.
authors.forename: Hecui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Horticulture and Landscape Architecture, Southwest University, Chongqing, 400716, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Lian
authors.affiliation: Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, 45267-0524, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Converse
authors.affiliation: College of Agronomy and Biotechnology, Southwest University, Chongqing, 400716, China. zhuliquan@swu.edu.cn.
authors.forename: Liquan
authors.initials: L
authors.lastname: Zhu
mesh_terms: D051186:Armadillo Domain Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D051186:Armadillo Domain Proteins; D010940:Plant Proteins; D008666:Metalloendopeptidases
keywords: Armadillo repeat containing 1; Brassica oleracea; Exocyst 70 A1; Interaction; Recognition motif