Cloning and characterization of CroMATE1, a novel MATE-type transporter from the medicinal plant Catharanthus roseus (L.)

Catharanthus roseus (L.) G. Don accumulates the dimeric terpenoid indole alkaloids (TIAs) vincristine and vinblastine, two of the most potent anticancer natural agents discovered to date. The low abundance of these compounds in the plant, together with their pharmacological importance, has made C. roseus one of the most studied medicinal plants. To date, the leaves of the plant remain the only source of the anticancer TIAs, since efforts to achieve the chemical synthesis or in vitro production, in attempt to enhance the productivity of the process, have been unsuccessful. The inherent complexity of the TIA biosynthesis pathway has been gradually unveiled. However, the existence of many enzymatic steps and the high compartmentalization of the process, involving different types of cells/organelles and therefore several transmembrane transport events, have hindered the possibility for manipulation of this biosynthetic pathway. Recently, it has become clear that the study of the mechanisms and mediators of the transmembrane transport steps of metabolic pathways provides an essential complement to the conventional metabolic engineering strategies aiming at enhancing the productivity of plant natural products (Yazaki, Sugiyama et al. 2007). The so-called multidrug transporters have been emerging as valuable candidates for the intraand inter-cellular transmembrane transport steps of secondary metabolites in plants, a role that could fill the gaps and clarify the flux mechanisms of TIAs and precursors in C. roseus. Within the multidrug transporters group, the MATE (multidrug and toxic compound extrusion) secondary transporters family is particularly abundant in plants and has already been shown to be implicated in the transport of several secondary compounds in plants. Namely, tobacco MATE-type transporters involved in the vacuolar transport of the alkaloid nicotine by a mechanism of proton-antiport were already characterized. Prior to this work, the vacuolar accumulation of TIAs in C. roseus was shown to be also mediated by a proton-antiport mechanism, which supports the potential involvement of a MATE-type transporter in the process (Carqueijeiro, Noronha et al. 2013). The work hereby described includes the isolation and the computational and experimental-based characterization of a C. roseus MATE-type candidate gene, CroMATE1, which codifies a protein previously detected with high abundance in the vacuole and tonoplast proteomes from C. roseus leaves, thus constituting an obvious candidate to the transport of TIAs into the vacuole. Based upon the sequences of the peptides identified in the proteomic study, it was possible to retrieve the full-length CroMATE1 coding sequence from C. roseus transcriptomic databases. By using such 4 FCUP Cloning and characterization of CroMATE1, a novel MATE-type transporter from the medicinal plant Catharanthus roseus (L.) G. Don information, the isolation, cloning and sequencing of CroMATE1 were successfully achieved. In a consistent fashion, the computational predictive analyses resulted in the identification of a diverse array of characteristics inherent to the coding sequence of CroMATE1, as well as to the respective protein, unveiling the identity of this candidate, the occurring co-regulation with genes involved in the TIA biosynthesis, and a potential vacuolar localization. This localization was experimentally confirmed via the transient transformation of C. roseus mesophyll protoplasts with fusions of CroMATE1 and a green fluorescent protein (GFP), which unequivocally showed the sorting of the transporter to the tonoplast. This result validates the informatics predictions previously performed and confirms the proteomic study of vacuoles, reinforcing the role of CroMATE1 as candidate for the vacuolar transport of TIAs in C. roseus. With the tools developed in this work it should be possible to determine experimentally the function of CroMATE1 in the near future. If the CroMATE1 involvement in the intra-vacuolar accumulation of C. roseus alkaloids is confirmed, this will be an important contribution towards the possibility of manipulating the C. roseus TIA pathway to obtain higher levels of the anticancer alkaloids.