Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters.
We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1.
Coady
M J
MJ
Groupe de Recherche en Transport Membranaire, Université de Montréal, Montreal, Que., Canada. coady@ere.umontreal.ca
Jalal
F
F
Bissonnette
P
P
Cartier
M
M
Wallendorff
B
B
Lemay
G
G
Lapointe
J
J
D019325:3-O-Methylglucose / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018076:DNA, Complementary; D005690:Galactose / Q000378:metabolism / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007294:Inositol / Q000378:metabolism / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins; D008757:Methylglucosides / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010695:Phlorhizin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D051269:Sodium-Glucose Transporter 1; D027981:Symporters; D019325:3-O-Methylglucose; C073884:SLC5A3 protein, human; D007294:Inositol; C027020:methylglucoside; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose; D005690:Galactose