Biology subjects

Beech, R. N.

Publications and source records attributed to Beech, R. N..

2 recordsLinked to original sources

bioRxiv2023

In vivo characterization of a secologanin transporter from Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel of in vivo biochemical characterization using the Xenopus laevis oocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, both in planta and in the X. laevis system. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasis in planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs. SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter in Catharanthus roseus, as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.

plant biology↗

bioRxiv2022

Reconstitution of an N-AChR from Brugia malayi

Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal current to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to maximal response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

molecular biology↗