The hepatocyte glucose-6-phosphatase subcomponent T3: its relationship to GLUT2.
Glucose-6-phosphatase (G6Pase) is a multiple protein complex in the endoplasmic reticulum (ER) that includes a mechanism (known as T3) for glucose exit from the ER to the cytosol. The molecular identity of T3 is not known. T3 has been shown to be functional in the absence of GLUT2, indicating that it is not GLUT2. Here we found a 55-kDa protein in high-density microsomal fraction (HDM) of rat hepatocytes that is recognized by polyclonal GLUT2 antibody raised against the GLUT2 C-terminal 14-amino-acid-sequence peptide. HDM contained calnexin but no integrin-beta1 or Na/K ATPase in Western blotting. Significant GLUT2 immunoreactivity was colocalized with colligin, an ER marker, in confocal microscopy. Furthermore, the 55-kDa protein in HDM was labeled with a covalently reactive, impermeable glucose transporter substrate, 1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoyl-benzoate (B3GL) when hepatocyte homogenates, but not intact cells, were labeled. In addition glucose efflux from HDM vesicles was sensitive to B3GL treatment in a dose-dependent manner. Based on these findings, we suggest that T3 may be a novel facilitative glucose transporter that is highly homologous to GLUT2 in the C-terminal sequence, thus cross-reacting with the GLUT2 antibody. The finding will be useful in molecular identification and cloning of T3.
Hah
Jong Sik
JS
The Biophysics Laboratory, Veterans Administration Medical Center, Buffalo, NY 14215, USA.
Ryu
Jiwon
J
Lee
Wan
W
Jung
Chan Y
CY
Lachaal
Mohsen
M
D000818:Animals; D001565:Benzoates; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism; D003847:Deoxyglucose / Q000031:analogs & derivatives*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D051273:Glucose Transporter Type 2; D005952:Glucose-6-Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D006023:Glycoproteins; D022781:Hepatocytes / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008297:Male; D008861:Microsomes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D001565:Benzoates; D002352:Carrier Proteins; D051273:Glucose Transporter Type 2; D006023:Glycoproteins; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D020033:Protein Isoforms; C046289:colligin; C076517:1,3-bis(3-deoxyglucopyranose-3-yloxy)-2-propyl-4-benzoylbenzoate; D003847:Deoxyglucose; D005952:Glucose-6-Phosphatase; D005947:Glucose