TABLE 1.
Zero-trans and hetero-exchange 2-DG uptake by GLUT1Myc-GLUT4Myc chimeras
ZT and HE uptakes were measured for GLUT1Myc in every assay. This table reports the GLUT1Myc data as a global mean ± S.E. for a minimum of n = 30 assays. The range observed in these assays for zero-trans uptake was 39.2 ± 5.36 to 185 ± 18.8 fmol/μg/min. The range observed for HE:ZT was 1.48 ± 0.11 to 2.3 ± 0.31.
| Chimeraa | Residuesb | ZT uptakec | Fold-stimulation during hetero-exchange (HE) HE:ZTd | Trans-acceleratione |
|---|---|---|---|---|
| fmol/μg/min | p value | |||
| GLUT1Myc | 1–492 | 117.2 ± 14.9 | 1.80 ± 0.15 | Y |
| p ≤ 0.001 | ||||
| GLUT4Myc | 1–509 | 42.0 ± 6.8 | 0.96 ± 0.08 | N |
| GLUT4Myc-3x | 1–509 | 77.4 ± 7.4 | 1.04 ± 0.45 | N |
| 44(1)11 | G4 1–223 | 35.4 ± 4.6 | 0.94 ± 0.17 | N |
| G1 208–492 | ||||
| 11(1)44 | G1 1–266 | 66.4 ± 7.4 | 1.90 ± 0.21 | Y |
| G4 283–509 | p ≤ 0.001 | |||
| 44(4)11 | G4 1–282 | 40.3 ± 9.0 | 0.96 ± 0.18 | N |
| G1 267–492 | ||||
| 11(4)44 | G1 1–207 | 35.3 ± 7.3 | 2.00 ± 0.31 | Y |
| G4 224–509 | p ≤ 0.01 | |||
| 1444 | G1 1–119 | 103.0 ± 7.0 | 1.10 ± 0.11 | N |
| G4 136–509 | ||||
| 4111 | G4 1–135 | 47.3 ± 5.9 | 1.70 + 0.21 | Y |
| G1 120–492 | p ≤ 0.01 | |||
| 1411 | G1 1–119; 208–492 | 146.1 ± 10.2 | 1.00 ± 0.13 | N |
| G4 136–223 | ||||
| 4144 | G4 1–135; 224–509 | 61.6 ± 8.1 | 1.80 ± 0.09 | Y |
| G1 120–207 | p ≤ 0.00001 | |||
| GLUT4Myc (4,5 G1) | G4 1–135; 203–509 | 60.1 ± 4.5 | 0.65 ± 0.15 | N |
| G1 120–186 | ||||
| GLUT4Myc (5,6 G1) | G4 1–166; 224–509 | 38.1 ± 3.9 | 1.90 ± 0.14 | Y |
| G1 151–207 | p ≤ 0.00001 | |||
| GLUT4Myc (5, G1) | G4 1–166; 203–509 | 53.2 ± 5.7 | 0.57 ± 0.09 | N |
| G1 151–186 | ||||
| GLUT4Myc (6, G1) | G4 1–203; 224–509 | 25.8 ± 6.2 | 1.80 ± 0.23 | Y |
| G1 187–207 | p ≤ 0.01 | |||
| GLUT1Myc (6, G4) | G1 1–186; 208–492 | 168.3 ± 12.5 | 1.10 ± 0.11 | N |
| G4 203–223 | ||||
| GLUT1Myc SIIFI 191–195 GLTVL | G1 1–190; 196–492 | 162.4 ± 7.9 | 1.30 ± 0.05 | Y |
| G4 208–212 | p ≤ 0.001 | |||
| GLUT1Myc CIV 202–204 LVL | G1 1–201; 205–492 | 64.1 ± 7.2 | 2.20 ± 0.18 | Y |
| G4 218–220 | p ≤ 0.00001 | |||
| GLUT4Myc GLTVL 208–212 SIIFI | G4 1–207; 213–509 | 55.8 ± 4.9 | 0.59 ± 0.09 | N |
| G1 191–195 | ||||
| GLUT4Myc LVL 218–220 CIV | G4 1–217; 221–509 | 92.1 ± 5.1 | 0.97 ± 0.05 | N |
| G1 202–204 |
a The chimeras employed in this study were constructed using two backbones: GLUT1Myc (wt GLUT1 residues 1–492 with a c-Myc epitope (EQKLISEEDL) inserted between residues 55 and 56) and GLUT4Myc-3x (wt GLUT4 residues 1–509 in which Phe-5, Leu-489, and Leu-490 is each mutagenized to Ala, and where a c-Myc epitope (EQKLISEEDL) inserted between residues 72 and 73). All residue numbering ignores the inserted c-Myc sequence. Chimera nomenclature is described under “Results” and in the legend to Fig. 1.
b The sequence composition of chimeras is described as fusions of GLUT1Myc (G1) and GLUT4Myc-3x (G4) sequence in which G1 and G4 sequence numbering ignores the inserted c-Myc epitope.
c ZT of 100 μm 2-DG (fmol/μg of protein/min) from medium containing 40 mm 3-MG was measured in transfected HEK cells depleted of intracellular sugar. Values are reported as mean ± S.E. for a minimum of n = 3 assays and are background-corrected for 2-DG uptake measured in non-transfected cells (41 ± 4 fmol/μg of protein/min).
d Stimulation of 2-DG uptake observed under hetero-exchange conditions (extra- and intracellular [3-MG] = 40 mm) was determined as the ratio of hetero-exchange (HE) 2-DG uptake to ZT uptake (fmol/μg/min). Values are reported as mean ± S.E. for a minimum of n = 3 assays.
e Trans-acceleration is absent (N) when HE:ZT is not significantly greater than 1. Trans-acceleration is present (Y) when HE:ZT is significantly greater than 1. Significance was determined using an unpaired, two-tailed Student's t test.