Specific residues in a purine transporter are critical for dimerization, ER exit, and function

Anezia Kourkoulou; Pothos Grevias; George Lambrinidis; Euan Pyle; Mariangela Dionysopoulou; Argyris Politis; Emmanuel Mikros; Bernadette Byrne; George Diallinas

doi:10.1534/genetics.119.302566

Specific residues in a purine transporter are critical for dimerization, ER exit, and function

Anezia Kourkoulou, Pothos Grevias, George Lambrinidis, Euan Pyle, Mariangela Dionysopoulou, Argyris Politis, Emmanuel Mikros, Bernadette Byrne, George Diallinas^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

9 Citations (Scopus)

Abstract

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

Original language	English
Pages (from-to)	1357-1372
Number of pages	16
Journal	Genetics
Volume	213
Issue number	4
DOIs	https://doi.org/10.1534/genetics.119.302566
Publication status	Published - 1 Jan 2019

Keywords

Aspergillus nidulans
NAT
Nucleobase
RSNBT1
UapA

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title = "Specific residues in a purine transporter are critical for dimerization, ER exit, and function",

abstract = "Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.",

keywords = "Aspergillus nidulans, NAT, Nucleobase, RSNBT1, UapA",

author = "Anezia Kourkoulou and Pothos Grevias and George Lambrinidis and Euan Pyle and Mariangela Dionysopoulou and Argyris Politis and Emmanuel Mikros and Bernadette Byrne and George Diallinas",

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doi = "10.1534/genetics.119.302566",

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T1 - Specific residues in a purine transporter are critical for dimerization, ER exit, and function

AU - Kourkoulou, Anezia

AU - Grevias, Pothos

AU - Lambrinidis, George

AU - Pyle, Euan

AU - Dionysopoulou, Mariangela

AU - Politis, Argyris

AU - Mikros, Emmanuel

AU - Byrne, Bernadette

AU - Diallinas, George

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

AB - Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

KW - Aspergillus nidulans

KW - NAT

KW - Nucleobase

KW - RSNBT1

KW - UapA

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DO - 10.1534/genetics.119.302566

M3 - Article

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AN - SCOPUS:85075985050

SN - 0016-6731

VL - 213

SP - 1357

EP - 1372

JO - Genetics

JF - Genetics

IS - 4

ER -

Specific residues in a purine transporter are critical for dimerization, ER exit, and function

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Keywords

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