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 t he beta cell glucose transporter GLUT2 was investigated in isolated is lets and insulinoma cells. Using a trypsin sensitivity assay to measur e 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, reexpress ion of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pu lse-labeling experiments combined with glycosidase treatment and the t rypsin sensitivity assay. We determined that transport from the endopl asmic 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 requi red a similar half-time. When added at the start of a pulse-labeling e xperiment, brefeldin A prevented exit of GLUT2 from the endoplasmic re ticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22-degr ees-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 surfa ce expression in beta cells is via the constitutive pathway, that tran sport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.