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).
REFERENCES
1. Brown D.
The ins and outs of aquaporin-2 trafficking. Am J Physiol Renal
Physiol 284:
F893-F901, 2003.
2. Bryant NJ, Govers R, and James DE. Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 4: 267-277, 2002.
3. Calhoun BC and Goldenring JR. Two Rab proteins, vesicle-associated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles. Biochem J 325: 559-564, 1997.[ISI][Medline]
4. Crane RK. Hypothesis for mechanism of intestinal active transport of sugar. Fed Proc 21: 891-895, 1962.[ISI]
5. Ellinger I, Klapper H, Courtoy PJ, Vaerman JP, and Fuchs R. Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: analysis by free-flow electrophoresis. Electrophoresis 23: 2117-2129, 2002.[ISI][Medline]
6. Hannig K and Heidrich HG. The use of continuous preparative free-flow electrophoresis for dissociating cell fractions and isolation of membranous components. Methods Enzymol 31: 746-761, 1974.[Medline]
7. Heidrich HG,
Kinne R, Kinne-Saffran E, and Hannig K. The polarity of the proximal
tubule cell in rat kidney. Different surface charges for the brush-border
microvilli and plasma membranes from the basal infoldings. J Cell
Biol 54: 232-245,
1972.
8. Hirsch JR, Loo
DD, and Wright EM. Regulation of Na+/glucose cotransporter
expression by protein kinases in Xenopus laevis oocytes. J
Biol Chem 271:
14740-14746, 1996.
9. Ikeda TS, Hwang ES, Coady MJ, Hirayama BA, Hediger MA, and Wright EM. Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine. J Membr Biol 110: 87-95, 1989.[ISI][Medline]
10. Kinne R, Heidrich HG, Hannig K, and Kinne-Saffran E. Separation of apical and basal cell sites from at kidney cortex. Pflügers Arch 332, Suppl 332: R29, 1972.
11. Kipp H, Khoursandi S, Scharlau D, and Kinne RKH. More than apical: distribution of SGLT1 in Caco-2 cells. Am J Physiol Cell Physiol 285: C737-C749, 2003.
12. Krivánková L and Bocek P. Continuous free-flow electrophoresis. Electrophoresis 19: 1064-1074, 1998.[ISI][Medline]
13. Schmid SL, Fuchs R, Male P, and Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell 52: 73-83, 1988.[ISI][Medline]
14. Schultz SG and
Zalusky R. Ion transport in isolated rabbit ileum. II. The interaction
between active sodium and active sugar transport. J Gen
Physiol 47:
1043-1059, 1964.
15. Wright EM.
Renal Na+-glucose cotransporters. Am J Physiol Renal
Physiol 280:
F10-F18, 2001.
16. Wright EM, Turk E, and Martin MG. Molecular basis for glucose-galactose malabsorption. Cell Biochem Biophys 36: 115-121, 2002.[ISI][Medline]
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |