Members of the Nitrate transporter 1/Peptide transporter Family (NPF) have been known for almost 30 years; still most aspects regarding the family, its substrates and its mechanism of transport remain largely unexplored. Although not necessarily with an associated phenotype, substrates are known for approximately half of the 53 Arabidopsis thalianaNPFproteins,which are the far most studied NPFproteins. The substrate specificity of NPFtransporters is a puzzle i.e.many NPF members display multi specificity and many members may recognize the some of the same substrates, especially among membrane permeable phytohormones. The crystal structure of a single NPF member has been solvedin a single conformation, but the mechanistic description which itcould facilitate is almost completely absent.In this dissertation, Iextend the knowledge onNPF transporters onthree facets; substrates, substrate specificity determinants and transport mechanism. Due to thehithertonear absence ofstructural and mechanistic investigations on NPFproteins, traits previously observed in distant NPF homologues from other branches oftheMajorFacilitator Superfamily,which apparently are shared with NPFmembers,are presented(ManuscriptI). By a phylogenic approach,differentially conserved amino acid positions that are involved in substrate dependent coupling to the proton gradientin the NPF glucosinolatetransportersubcladewereidentified(ManuscriptV). The development of an optimizedassay with the phytohormones known as gibberellic acids (GAs) and the use of thisassay to reduce the number of bona fideGA transportersare described. From the dataset,conserved amino acid positions in thesubstratebinding pocketof NPF proteins that may reflect the abilityof some NPFGAtransporters to recognize and transport GAsare presented(ManuscriptII). The still largeamount ofNPF GA transporters without a phenotypemay reflect functional redundancyamong NPF transporters, which may bethe case for two close paralogous transporters. However, in vitrothese two transportersdo displaymechanistic differencesthat suggestsub-functionalization in planta(ManuscriptIII). Additionally, the first NPF GA transporter capable of mediating GA effluxis presented(ManuscriptIV).Furthermore, two articleswere co-authored and published which areunrelated to NPF transport mechanism(Article I and II).
The results presentedhereprovide a foundation for future studies on NPF proteins and their transport mechanism.Empathyis put on how NPFtransporters,some of theirsubstratethemselves and the coupling mechanism are influenced by protons, thus protons have the potential to play an enormous rolein the transport events mediated by NPF proteins.