title: Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.
abstract: Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein-protein interaction.
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Ananya
authors.initials: A
authors.lastname: Marik
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Haraprasad
authors.initials: H
authors.lastname: Naiya
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Madhumanti
authors.initials: M
authors.lastname: Das
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Gairik
authors.initials: G
authors.lastname: Mukherjee
authors.affiliation: Department of Microbiology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Soumalee
authors.initials: S
authors.lastname: Basu
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India.
authors.forename: Chinmay
authors.initials: C
authors.lastname: Saha
authors.affiliation: Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A and 2B Raja S.C Mullick Road, Jadavpur, Kolkata, 700032, India.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Chowdhury
authors.affiliation: Department of Physical Chemistry, Indian Association for the Cultivation of Science, 2A and 2B Raja S.C Mullick Road, Jadavpur, Kolkata, 700032, India.
authors.forename: Kankan
authors.initials: K
authors.lastname: Bhattacharyya
authors.affiliation: Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India. aninditaseal@gmail.com.
authors.forename: Anindita
authors.initials: A
authors.lastname: Seal
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D009149:Mustard Plant / Q000235:genetics* / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D013879:Thioredoxins
keywords: Metal stress; NRAMP; Protein–protein interaction; Redox; Thioredoxin