Expression and purification of a functional uric acid-xanthine transporter (UapA).
The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Delta deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a approximately 5-fold increase in uptake of [(3)H]-xanthine compared to the pep4Delta deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Delta deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein.
Division of Molecular Biology, Imperial College London, South Kensington, London SW7 2AZ, UK.
James
J
Leung
Mayia
M
Karachaliou
Claudia
C
Alves
George
G
Diallinas
Bernadette
B
Byrne
D001233:Aspergillus nidulans / Q000235:genetics*; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015854:Up-Regulation
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C083852:UAPA protein, Aspergillus nidulans; D049452:Green Fluorescent Proteins