DISCRETE STRUCTURAL DOMAINS DETERMINE DIFFERENTIAL ENDOPLASMIC-RETICULUM TO GOLGI TRANSIT TIMES FOR GLUCOSE-TRANSPORTER ISOFORMS

The rate of movement of the glucose transporter isoforms Glut1 and Glu t4 from the endoplasmic reticulum (ER) to the Golgi apparatus was inve stigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell lin e that naturally expresses both transporter isoforms. Despite their hi gh degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t(1/2) values for ER to Golgi transit fo r Glut1 and Glut4 were < 1 and 24 h, respectively, in oocytes and simi lar to 5 and 20 min, respectively, in 3T3-L1 adipocytes. Pulse-chase i n conjunction with sucrose density gradient analysis revealed that the rate-limiting step in the ER to Golgi processing of Glut4 was exit fr om the ER and not retention in an early Gels compartment, We analyzed the biosynthesis of Glut1/Glut4 chimeric transporters in Xenopus oocyt es in order to determine whether specific domains in Glut1 and Glut4 w ere responsible for their distinct transit times. The first exofacial glycosylated loop and the cytoplasmic carboxyl-terminal domain of Glut 4 were crucial for its delayed exit from the ER. The first transmembra ne, the first exofacial, and the cytoplasmic COOH-terminal domains of Glut1 were largely responsible for Glut1's rapid processing in the ER Some of the chimeric transporters were not fully processed. Approximat ely 50% of chimeric molecules containing the cytoplasmic COOH-terminal domain of Glut1 and either the first transmembrane or first exofacial domain of Glut4 were retained in early Golgi compartments and prevent ed from complete maturation, Normal processing of these chimeras was a chieved by replacing the cytoplasmic COOH-terminal domain of Glut1 wit h that of Glut4. These data suggest that amino acid residues within th e glycosylated exofacial loop and the cytoplasmic COOH terminus partic ipate in a rate-limiting step in the folding of both Glut1 and Glut4 o r could act as transient ER retention signals. Additionally, these res ults show that even chimeric molecules constructed from two highly hom ologous proteins can exhibit aberrant folding and post-translational p rocessing.