GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.
The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.
Thorens
B
B
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Gérard
N
N
Dériaz
N
N
D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology*; D051273:Glucose Transporter Type 2; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D003517:Cyclopentanes; D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D014357:Trypsin