Membrane Topology of the Human Na+/Glucose Cotransporter SGLT1

Turk, Eric; Kerner, Cynthia J.; Lostao, M. P.; Wright, Ernest M.

doi:10.1074/jbc.271.4.1925

Cited by 160 publications

(146 citation statements)

References 39 publications

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“…Furthermore, these studies indicate that eukaryotic membrane proteins do not require cholesterol for activity-a steroid is missing among the E. coli membrane lipids-although the presence of cholesterol in the proteoliposomes increases the initial rates of sugar uptake by about 40%. This report also supports the observation that N-glycosylation is not required for hSGLT1 activity (28), because bacteria do not possess glycosylation machinery.…”

Section: Table 1 Kinetics Of Hsglt1 In Different Systemssupporting

confidence: 88%

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

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Wright

2002

Proc. Natl. Acad. Sci. U.S.A.

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Large-scale purification of recombinant human membrane proteins represents a rate-limiting step toward the understanding of their role in health and disease. There are only four mammalian membrane proteins of known structure, and these were isolated from natural sources (see http:͞͞www.mpibp-frankfurt.mpg.de͞michel͞ public͞memprotstruct.html). In addition, genetic diseases of membrane proteins are frequently caused by trafficking defects, and it is enigmatic whether these mutants are functional. Here, we report the employment of Escherichia coli for the functional expression, purification, and reconstitution of a human membrane protein, the human Na ؉ ͞glucose cotransporter (hSGLT1). The use of an E. coli mutant defective in the outer membrane protease OmpT, incubation temperatures below 20°C, and transcriptional regulation from the lac promoter͞operator are crucial to reduce proteolytic degradation. Purification of a recombinant hSGLT1 through affinity chromatography yields about 1 mg of purified recombinant hSGLT1 per 3 liters of cultured bacterial cells. Kinetic analysis of hSGLT1 in proteoliposomes reveals that a purified recombinant transporter, which is missorted in eukaryotic cells, retains full catalytic activity. These results indicate the power of bacteria to manufacture and isolate human membrane proteins implicated in genetic diseases.I ntegral membrane proteins such as channels and transporters are essential for all living organisms by mediating the passage of ions and molecules across membranes. The understanding of how membrane proteins work in health and disease has been restricted largely because of the lack of purified protein. Most membrane proteins are present at low concentrations in their native tissue, and their overexpression is a prerequisite for purification. The use of eukaryotic expression systems has illuminated mechanistic features of human membrane proteins, but these are unsuitable for their large-scale purification (1, 2). In addition, genetic disorders of membrane proteins are frequently caused by improper trafficking in cells, and it is not known whether missorted proteins retain their catalytic activity (3). To obtain structural information on membrane proteins, prokaryotic homologues have been most amenable because they can often be expressed in bacteria in large quantities (4). However, the inexpensive and less time-consuming overexpression of animal and human integral proteins of the plasma membrane has not been applicable, and there are only a few examples in which eukaryote membrane proteins are functional when expressed in prokaryotic cells (5-11). Here, we report the employment of Escherichia coli for the expression, purification, and reconstitution of a fully functional recombinant human sodium͞glucose transporter (hSGLT1)-a central protein for the homeostasis of glucose, salt, and water (12-14). Our strategy is based in part on our group's previous success in purifying functional transporters from bacteria (15, 16). Experimental ProceduresGenetic Engineering. A casset...

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Section: Table 1 Kinetics Of Hsglt1 In Different Systemssupporting

confidence: 88%

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

Quick

Wright

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…However, assuming that at least one of the two potential N-glycosylation sites (NXS/T) at the Cterminal end of the protein (Asn 586 and Asn 598 ) is actually used, both the N terminus and the C terminus would have to be localized extracellularly according to all these models. Such an orientation has been suggested previously for the Na ϩ -D-glucose cotransporter SGLT1 (30). There are several potential phosphorylation sites for protein kinase C and casein kinase II (cf.…”

Section: Fnadc-3 Sequence and Comparison With Related Data Basesupporting

confidence: 63%

Expression Cloning and Characterization of a Novel Sodium-Dicarboxylate Cotransporter from Winter Flounder Kidney

Steffgen

Burckhardt

Langenberg

et al. 1999

Journal of Biological Chemistry

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A cDNA coding for a Na ؉ -dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevis oocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (K m ) of fNaDC-3 for succinate of 30 M resembles that of Na ؉ -dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na ؉

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“…Based on theoretical considerations and data from glycosylation mutants, position 166 is hypothesized to be part of a short 16 amino acid loop connecting transmembrane helices IV and V of SGLT1 (21). To date, the only experimental data ascribing function to any part of this loop comes from a study by Panayotova-Heiermann, et al (6) in which the aspartic acid at position 176 (located at the transmembrane V end of the loop) was mutated to an alanine.…”

Section: Discussionmentioning

confidence: 99%

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

Lo¹,

Silverman

1998

Journal of Biological Chemistry

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An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit sodium/glucose transporter (rSGLT1). When expressed in Xenopus laevis oocytes, this mutant transporter (A166C rSGLT1) demonstrates a significantly lower apparent affinity for ␣-methyl glucoside (␣MG) compared with the wild-type transporter (apparent K m ‫؍‬ 0.8 versus 0.15 mM). Using the two-electrode voltage clamp technique, transient currents have also been measured, and for the mutant transporter, the transients induced by large depolarizations exhibit longer time constants than those for wild type. Moreover, the substitution of Ala-166 with a cysteine allows the sulfydryl specific reagent, methanethiosulfonate ethylamine (MTSEA), to react with and alter the function of the transporter. Whereas the wild-type transporter is unaffected by reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced by 1 mM ␣MG inhibited 83% within a minute of exposure to MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant after MTSEA exposure demonstrate much shorter time constants than before while the total amount of charge transferred is only slightly diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.

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Membrane Topology of the Human Na+/Glucose Cotransporter SGLT1

Cited by 160 publications

References 39 publications

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

Employing Escherichia coli to functionally express, purify, and characterize a human transporter

Expression Cloning and Characterization of a Novel Sodium-Dicarboxylate Cotransporter from Winter Flounder Kidney

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

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