RESUMO
Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling.
Assuntos
Proteínas de Cloroplastos , Sistema Enzimático do Citocromo P-450 , Proteínas de Membrana , Nicotiana , Nitrilas/metabolismo , Proteínas de Plantas , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Tilacoides , Proteínas de Cloroplastos/biossíntese , Proteínas de Cloroplastos/genética , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Tilacoides/genética , Tilacoides/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Transforming growth factor ß (TGF-ß) signaling is regulated by clathrin-dependent endocytosis (CDE) for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part of the cilium is a site for CDE. We report here that TGF-ß receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-ß stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF-ß-mediated signaling at the cilium, and TGF-ß signaling and CDE activity are reduced at stunted primary cilia in Tg737orpk fibroblasts. Similarly, TGF-ß signaling during cardiomyogenesis correlated with accumulation of TGF-ß receptors and activation of SMAD2/3 at the ciliary base. Our results indicate that the primary cilium regulates TGF-ß signaling and that the ciliary pocket is a compartment for CDE-dependent regulation of signal transduction.
