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 Glut4 from the endoplasmic reticulum (ER) to the Golgi apparatus was investigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell line that naturally expresses both transporter isoforms. Despite their high degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t1/2 values for ER to Golgi transit for Glut1 and Glut4 were < 1 and 24 h, respectively, in oocytes and approximately 5 and 20 min, respectively, in 3T3-L1 adipocytes. Pulse-chase in conjunction with sucrose density gradient analysis revealed that the rate-limiting step in the ER to Golgi processing of Glut4 was exit from the ER and not retention in an early Golgi compartment. We analyzed the biosynthesis of Glut1/Glut4 chimeric transporters in Xenopus oocytes in order to determine whether specific domains in Glut1 and Glut4 were responsible for their distinct transit times. The first exofacial glycosylated loop and the cytoplasmic carboxyl-terminal domain of Glut4 were crucial for its delayed exit from the ER. The first transmembrane, 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. Approximately 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 prevented from complete maturation. Normal processing of these chimeras was achieved by replacing the cytoplasmic COOH-terminal domain of Glut1 with that of Glut4. These data suggest that amino acid residues within the glycosylated exofacial loop and the cytoplasmic COOH terminus participate in a rate-limiting step in the folding of both Glut1 and Glut4 or could act as transient ER retention signals. Additionally, these results show that even chimeric molecules constructed from two highly homologous proteins can exhibit aberrant folding and post-translational processing.
Hresko
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RC
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Murata
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H
Marshall
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BA
Mueckler
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M
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D009124:Muscle Proteins; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D014982:Xenopus laevis
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495678:Slc2a1 protein, mouse; C495679:Slc2a1 protein, rat; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat