Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family.
NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.
Xie
Hao
H
Astbury Centre for Structural Molecular Biology University of Leeds, LS2 9JT Leeds, UK.
Patching
Simon G
SG
Gallagher
Maurice P
MP
Litherland
Gary J
GJ
Brough
Adrian R
AR
Venter
Henrietta
H
Yao
Sylvia Y M
SY
Ng
Amy M L
AM
Young
James D
JD
Herbert
Richard B
RB
Henderson
Peter J F
PJ
Baldwin
Stephen A
SA
D000241:Adenosine / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D009865:Oocytes / Q000737:chemistry; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D014529:Uridine / Q000032:analysis / Q000378:metabolism; D014981:Xenopus
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; C098551:NupG protein, E coli; D011994:Recombinant Proteins; D000241:Adenosine; D014529:Uridine