Continuous flow electrophoresis for study of membrane protein compartments. Focus on "More than apical: distribution of SGLT1 in Caco-2 cells"

John Cuppoletti

Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05

THE UPTAKE OF GLUCOSE by the intestine is sodium dependent (4). The flow of glucose is accompanied by the movement of an equivalent number of sodium ions (14).

There are at least three human forms of sodium-dependent glucose transporters: SGLT1, SGLT2, and SGLT3 (15). SGLT1 was cloned from rabbit intestine (9), and when this protein was expressed in Xenopus laevis oocytes, D-glucose uptake rates across cells were increased by activation of protein kinase A and were also affected by protein kinase C in a reversible manner and over a time course of minutes. The increased transport rates in cells correlated with a change in the number of transporters and the surface area of the apical membrane (8), suggesting an important role of trafficking in regulation of this protein in the physiological state. Trafficking defects due to mutations of the protein also play an important role in some patients exhibiting glucose-galactose malabsorption disease (16).

The study by Kipp et al., the current article in focus (Ref. 11, see p. C737 in this issue), undertook to elucidate the nature of the intracellular pools of SGLT1 by using a model cell system for the intestine, Caco-2 cells grown under polarized conditions. They prepared antibodies to extracellular loops of SGLT1 to circumvent the possibility that intracellular binding partners of SGLT1 might interfere with antigenic sites on intracellular residues. These antibodies were used to immunolocalize SGLT1 in Caco-2 cells before and after permeabilization, demonstrating that SGLT1 existed in the apical membrane and in regions within the cells. In striking micrographs, SGLT1 was shown to be associated with microtubules, immediately suggesting a role for microtubules in regulation of D-glucose transport protein in the apical membrane. They then used free-flow electrophoresis to isolate plasma membranes, basolateral membranes, and early and late endosomes and then quantified the distribution of the SGLT1 in each of the fractions. SGLT1 was found to be associated with early endosomes. The ratio of distribution of SGLT1 was approximately two parts intracellular pool to one part apical membrane. Using pulse chase with [S35]methionine, they found that SGLT1 had a half-life of ~2.5 days and that cycloheximide treatment did not alter the relative distribution of SGLT1.

SGLT1 is another example of a membrane transporter whose activity is regulated by translocation between the intracellular vesicles and the plasma membrane (1, 2, 8). The article in focus clearly defines the intracellular pool involved in SGLT1 regulation and points out the value of continuous free-flow electrophoresis as a highly useful technique for the separation of biological membranes. Free-flow electrophoresis separates components of membrane suspensions when they are injected into a continuously flowing medium subjected to an electric field. Separation depends on the surface characteristics of the membranes under defined conditions. The separated membranes are then collected and subjected to functional, biochemical, or immunological analysis (6, 12).

One of the authors (R. Kinne) was a pioneer in the use of the technique, demonstrating that free-flow electrophoresis could be used to separate apical and basolateral membranes on the basis of differential charge (7, 10). The technique was used to demonstrate sub-populations of endosomes of different origins (13) and to separate subcellular organelles with different functions (5).

Continuous free-flow electrophoresis is a powerful method for study of membrane proteins that can be coupled with other techniques, such as immunopurification of membranes (3), for studies of membrane protein trafficking and studies of the proteomics of membrane protein regulation.


Address for reprint requests and other correspondence: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, Cincinnati, OH 45267-05 (E-mail: john.cuppoletti{at}uc.edu).

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