A method for expression cloning of transporter genes by screening yeast for uptake of radiolabelled substrate.
A method has been developed for the cloning of plasma membrane transporters by screening yeast transformed with a cDNA library for the accumulation of radiolabelled substrate. The applicability of the method is demonstrated by cloning the amino acid permease AAP1. A yeast mutant defective in proline uptake was transformed with an Arabidopsis thaliana cDNA library and plated on medium supplemented with L-[U-(14)C]proline. Yeast colonies accumulating radiolabelled proline were identified by autoradiography. The plasmids of these colonies were reintroduced into the yeast mutant and restoration of proline uptake was confirmed by L-[U-(14)C]proline uptake measurements. Whereas cloning of transporters by functional complementation requires that the substrate taken up is metabolized by yeast to promote growth, the method described here can be used to isolate transporters of substrates which are not metabolized. The method has great potential for the isolation of transporters of various substrates such as secondary plant products.
Wittstock
U
U
Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, and Centre of Molecular Plant Physiology (PlaCe), 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Chen
S
S
Halkier
B A
BA
D017360:Arabidopsis / Q000235:genetics*; D001692:Biological Transport; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D015723:Gene Library; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D026901:Membrane Transport Proteins; D011392:Proline