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oai:recercat.cat:2117/4464142026-02-11T06:25:49Zcom_2072_1033col_2072_452950

The 5-formyl-tetrahydrofolate proteome links folates with C/N metabolism and reveals feedback regulation of folate biosynthesis Li, Weichao Liang, Qiuju Mishra, Ratnesh Sánchez Muñoz, Raúl Wang, Huan Chen, Xin Van Der Straeten, Dominique Zhang, Chunyi Xiao, Youli Universitat Politècnica de Catalunya. Departament d'Enginyeria Agroalimentària i Biotecnologia Universitat Politècnica de Catalunya. HorPTA - Horticultura: producció, transformació i aprofitament Àrees temàtiques de la UPC::Enginyeria agroalimentària::Agricultura::Biotecnologia i millora genètica vegetal Folates Folate biosynthesis Folates are indispensable for plant development, but their molecular mode of action remains elusive. We synthesized a probe, “5-F-THF-Dayne,” comprising 5-formyl-tetrahydrofolate (THF) coupled to a photoaffinity tag. Exploiting this probe in an affinity proteomics study in Arabidopsis thaliana, we retrieved 51 hits. Thirty interactions were independently validated with in vitro expressed proteins to bind 5-F-THF with high or low affinity. Interestingly, the interactors reveal associations beyond one-carbon metabolism, covering also connections to nitrogen (N) metabolism, carbohydrate metabolism/photosynthesis, and proteostasis. Two of the interactions, one with the folate biosynthetic enzyme DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE 1 (AtDHFR-TS1) and another with N metabolism-associated glutamine synthetase 1;4 (AtGLN1;4), were further characterized. In silico and experimental analyses revealed G35/K36 and E330 as key residues for the binding of 5-F-THF in AtDHFR-TS1 and AtGLN1;4, respectively. Site-directed mutagenesis of AtGLN1;4 E330, which co-localizes with the ATP-binding pocket, abolished 5-F-THF binding as well as AtGLN1;4 activity. Furthermore, 5-F-THF was noted to competitively inhibit the activities of AtDHFR-TS1 and AtGLN1;4. In summary, we demonstrated a regulatory role for 5-F-THF in N metabolism, revealed 5-F-THF-mediated feedback regulation of folate biosynthesis, and identified a total of 14 previously unknown high-affinity binding cellular targets of 5-F-THF. Together, this sets a landmark toward understanding the role of folates in plant development. Postprint (published version) 2021-10-11 Article Li, W. [et al.]. The 5-formyl-tetrahydrofolate proteome links folates with C/N metabolism and reveals feedback regulation of folate biosynthesis. «Plant cell», 11 Octubre 2021, vol. 33, núm. 10, p. 3367-3385. 1040-4651 https://hdl.handle.net/2117/446414 10.1093/plcell/koab198 eng https://academic.oup.com/plcell/article/33/10/3367/6342425 Open Access 19 p. application/pdf