title: Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.
abstract: In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.
authors.affiliation: Department of Biophysical Sciences, State University of New York, Buffalo.
authors.forename: I
authors.initials: I
authors.lastname: Jo
authors.forename: J S
authors.initials: JS
authors.lastname: Hah
authors.forename: A L
authors.initials: AL
authors.lastname: Rampal
authors.forename: R
authors.initials: R
authors.lastname: Chakrabarti
authors.forename: A R
authors.initials: AR
authors.lastname: Paterson
authors.forename: J D
authors.initials: JD
authors.lastname: Craik
authors.forename: C E
authors.initials: CE
authors.lastname: Cass
authors.forename: C R
authors.initials: CR
authors.lastname: Zobel
authors.forename: C Y
authors.initials: CY
authors.lastname: Jung
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008561:Membrane Fusion; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D051381:Rats; D014357:Trypsin / Q000378:metabolism
chemical_list: D007328:Insulin; D009004:Monosaccharide Transport Proteins; D014357:Trypsin; D005947:Glucose