Substituted cysteine accessibility method analysis of human concentrative nucleoside transporter hCNT3 reveals a novel discontinuous region of functional importance within the CNT family motif (G/A)XKX3NEFVA(Y/M/F).
The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).
Slugoski
Melissa D
MD
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Ng
Amy M L
AML
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Yao
Sylvia Y M
SYM
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Lin
Colin C
CC
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Mulinta
Ras
R
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada.
Cass
Carol E
CE
Oncology, Membrane Protein Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
Baldwin
Stephen A
SA
Astbury Centre for Structural Molecular Biology, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Young
James D
JD
From the Departments of Physiology, Edmonton, Alberta T6G 2H7, Canada. Electronic address: james.young@ualberta.ca.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014529:Uridine / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
D026901:Membrane Transport Proteins; D009705:Nucleosides; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; D014529:Uridine