PhD defence: Nikolai Wulff

Title of thesis: Unravelling the Transport Mechanism of NPF Transporters​

Summary:​​​
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 thaliana NPF proteins, which are the far most studied NPF proteins. The substrate specificity of NPF transporters 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 solved in a single conformation, but the mechanistic description which it could facilitate is almost completely absent.
In this dissertation, I extend the knowledge on NPF transporters on three facets; substrates, substrate specificity determinants and transport mechanism. Due to the hitherto near absence of structural and mechanistic investigations on NPF proteins, traits previously observed in distant NPF homologues from other branches of the Major Facilitator Superfamily, which apparently are shared with NPF members, are presented (Manuscript I). By a phylogenic approach, differentially conserved amino acid positions that are involved in substrate dependent coupling to the proton gradient in the NPF glucosinolate transporter subclade were identified (Manuscript V). The development of an optimized assay with the phytohormones known as gibberellic acids (GAs) and the use of this assay to reduce the number of bona fide GA transporters are described. From the dataset, conserved amino acid positions in the substrate binding pocket of NPF proteins that may reflect the ability of some NPF GA transporters to recognize and transport GAs are presented (Manuscript II). The still large amount of NPF GA transporters without a phenotype may reflect functional redundancy among NPF transporters, which may be the case for two close paralogous transporters. However, in vitro these two transporters do display mechanistic differences that suggest sub-functionalization in planta (Manuscript III). Additionally, the first NPF GA transporter capable of mediating GA efflux is presented (Manuscript IV). Furthermore, two articles were co-authored and published which are unrelated to NPF transport mechanism (Article I and II).
The results presented here provide a foundation for future studies on NPF proteins and their transport mechanism. Empathy is put on how NPF transporters, some of their substrate themselves and the coupling mechanism are influenced by protons, thus protons have the potential to play an enormous role in the transport events mediated by NPF proteins.
Supervisor:​
Professor Hussam Nour-Eldin, Molecular Plant Biology
Assessment committee:​​
Professor Benoit Lacombe, CNRS Montpellier, France
Professor Christian Loew, CSSB Hamburg, Germany
Associate Professor Rosa Laura Lopez Marques, PLEN (chair)
Reception afterwards in R322 and R323​​