Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens

Minton, Janet A.; Rapp, Micah; Stoffer, Amanda; Schultes, Neil P.; Mourad, George

doi:10.1016/j.plaphy.2015.12.014

Cited by 10 publications

(5 citation statements)

References 37 publications

(42 reference statements)

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“…Fungal Fcy-like transporters have an entirely different and nonoverlapping specificity profile from that of the Fur transporters, being high-affinity H 1 symporters specific for cytosine, adenine, guanine, hypoxanthine or pyridoxine (Weber et al, 1990;Stolz and Vielreicher, 2003;Paluszynski et al, 2006;Vlanti and Diallinas, 2008;Krypotou et al, 2012). Plant NCS1 transporters are more similar to fungal Fur sequences but show a specificity profile overlapping that of fungal Fcy and Fur proteins; they transport adenine, guanine, allantoin and uracil (Mourad et al, 2012;Schein et al, 2013;Witz et al, 2012;Minton et al, 2016;Rapp et al, 2016). The two functionally known bacterial NCS1 transporters, CodB in Escherichia coli (Danielsen et al, 1992) and Mhp1 in Microbacterium liquefaciens (Weyand et al, 2008), are specific for cytosine and benzyl-hydantoin, respectively.…”

Section: Introductionmentioning

confidence: 99%

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

Sioupouli

Lambrinidis

Mikros

et al. 2016

Molecular Microbiology

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NCS1 proteins are H or Na symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.

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Section: Introductionmentioning

confidence: 99%

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

Sioupouli

Lambrinidis

Mikros

et al. 2016

Molecular Microbiology

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“…Yeast genomes do not contain loci for xanthine or uric acid transport. These yeast strains and expression plasmids have been successfully employed in heterologous expression experiments monitoring for the uptake of radiolabeled purines and pyrimidines for a variety of plant NCS1 and NCS2 loci for guanine, adenine, cytosine, hypoxanthine, xanthine, and uracil [48][49][50][51][52]. A time course monitoring the uptake of [ 3 H]adenine for strain RG191 harboring PlUacP (RG191/ pNS515) displays a saturation after 60 min (Fig.…”

Section: P Larvae Genome Contains a Locus With Similarities To Bacter...mentioning

confidence: 99%

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae

et al. 2018

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Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease‐like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter‐deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine K m 7.04 ± 0.18 μ m , and as with other bacterial and plant NCS 2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m‐chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.

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“…The APC superfamily includes the Nucleobase Cation Symporter 1 (NCS1) group, of which several fungal and plant transporters have been extensively studied at the genetic and functional level [27][28][29][30][31][32][33][34]. In particular, work performed in Aspergillus nidulans, a filamentous fungus that has developed to be a model system to study transporters [35][36][37], has unveiled important knowledge on the regulation of expression, subcellular trafficking, turnover, transport kinetics and substrate specificity of NCS1 transporters [27][28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit

Pyrris,

Papadaki,

Mikros

et al. 2024

Microb Cell

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FurE is a H+ symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus Aspergillus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic mutational analysis of TMS11-12 of FurE and show that two specific aromatic residues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE molecules oligomerize or partition in specific microdomains to achieve concentrative ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intramolecular chaperone for proper FurE folding, which seems to provide a structural code for FurE partitioning in ER-exit sites.

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Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens

Cited by 10 publications

References 37 publications

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit

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