Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis

We have identified and characterized three genes, SUT1, SUT2 and SUT3, that encode glucose transporters of the yeast Pichia stipitis. When expressed i n a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, all three proteins restored growth on glucose. Sequenci ng of the genes revealed open reading frames coding for 553 amino acids in the case of SUT1, and for 550 amino acids in the case of SUT2 and of SUT3, The derived protein sequences are closely related to one another, and show distinct sequence similarities to the S. cerevisiae hexose transporter fami ly and to monosaccharide transporters of other organisms. The Sut2 and Sut3 proteins are nearly identical and differ only in one amino acid. Determina tion of substrate specificities and kinetic parameters of the individual Su t proteins expressed in a S. cerevisiae hxt1-7 mutant revealed Sut1, Sut2 a nd Sut3 as glucose transporters with K-m values in the millimolar range. Th e proteins were also able to transport xylose and other monosaccharides, bu t with a considerably lower affinity. In P. stipitis, transcription of SUT1 was strongly induced by glucose and was independent of the oxygen supply. In contrast, SUT2 and SUT3 were only expressed under aerobic conditions, bu t independent of the carbon source. Cells disrupted for the SUT1 gene did n ot show any obvious growth phenotype, however low-affinity glucose uptake w as lost. Further investigations suggest that the Sut proteins constitute a subfamily of glucose transporters in P. stipitis, and that other and probab ly unrelated proteins exist additionally mediating high-affinity glucose an d xylose uptake.