Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine–uric acid transporter and an intrinsically misfolded polypeptide Emilia Krypotou a , Claudio Scazzocchio b,c , George Diallinas a,⇑ a Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece b Department of Microbiology, Imperial College, London SW7 2AZ, United Kingdom c Institut de Génétique et Microbiologie, Université Paris-Sud, France article info Article history: Received 21 November 2014 Accepted 21 January 2015 Available online 29 January 2015 Keywords: Purine Fungi Phylogeny Pseudogene Specificity abstract The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H + /xanthine–uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed. Ó 2015 Published by Elsevier Inc. 1. Introduction The ubiquitous Nucleobase-Ascorbate Transporter (NAT) family, also known as the Nucleobase-Cation Symporter 2 (NCS2) family, includes thousands of putative members in nearly all organisms in almost every phylum (Diallinas and Gournas, 2008; Papageorgiou et al., 2008; Frillingos, 2012). NAT proteins share 14 a-helical hydrophobic transmembrane segments (TMS) and cytoplasmic N- and C-terminal regions, as well as, significant over- all amino acid sequence identity (>24% among bacteria, fungi, plants or mammals) and conserved motifs. The two most conserved motifs, located in TMS1 (Gln-His) and TMS10 (Gln/ Glu/Pro-Asn-X-Gly-X 4 -Thr-Arg/Lys/Gly), serve as additional diag- nostic criteria for NAT transporters. These motifs are part of the substrate-binding site of NATs (Amillis et al., 2011; Karena and Frillingos, 2011; Kosti et al., 2012) Approximately 20 members of the NAT family have been func- tionally characterized to date (see Frillingos, 2012). Most of these are H + (bacteria, fungi, plants) or Na + (mammals) coupled symport- ers specific for the cellular uptake of xanthine, uric acid or uracil, and related analogues or drugs. Interestingly, in primates, NATs are solely specific for L-ascorbic acid (vitamin C) transport. Structure– function relationships of two NAT proteins, UapA from the ascomy- cete Aspergillus nidulans (Diallinas et al., 1998; Meintanis et al., 2000; Koukaki et al., 2005; Vlanti et al., 2006; Pantazopoulou and Diallinas, 2007; Papageorgiou et al., 2008; Kosti et al., 2010; Amillis et al., 2011) and XanQ from Escherichia coli (Georgopoulou et al., 2010; Mermelekas et al., 2010; Frillingos, 2012) have been studied in great detail. These studies have led to the identification of amino acids critical for substrate recognition and transport. The publication of the first crystal structure of a bacterial NAT member in 2011, the uracil transporter UraA from E. coli (Lu et al., 2011), together with genetic and biochemical studies in UapA and XanQ, led to a coherent view concerning structure–function relationships in NATs. An important novel concept arising from the studies on UapA is that http://dx.doi.org/10.1016/j.fgb.2015.01.009 1087-1845/Ó 2015 Published by Elsevier Inc. ⇑ Corresponding author. Fax: +30 (210)7274702. E-mail address: diallina@biol.uoa.gr (G. Diallinas). Fungal Genetics and Biology 75 (2015) 56–63 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi